PH0326 BCA蛋白浓度测定试剂盒使用手册

INSTRUCTIONSPierce® BCA Protein Assay Kit23225 Pierce BCA Protein Assay Kit, sufficient reagents for 500 test-tube or 5000 microplate assays 23227 Pierce BCA Protein Assay Kit, sufficient reagents for 250 test-tube or 2500 microplate assays Kit Contents:BCA Reagent A, 1000mL (in Product No. 23225) or 500mL (in Product No. 23227), containingsodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartrate in 0.1M sodiumhydroxideBCA Reagent B, 25mL, containing 4% cupric sulfateAlbumin Standard Ampules, 2mg/mL, 10 × 1mL ampules, containing bovine serum albumin (BSA)at 2mg/mL in 0.9% saline and 0.05% sodium azideStorage: Upon receipt store at room temperature. Product shipped at ambient temperature.Note: If either Reagent A or Reagent B precipitates upon shipping in cold weather or during long-termstorage, dissolve precipitates by gently warming and stirring solution. Discard any kit reagent thatshows discoloration or evidence of microbial contamination.Table of ContentsIntroduction (1)Preparation of Standards and Working Reagent (required for both assay procedures) (2)Test Tube Procedure (Sample to WR ratio = 1:20) (3)Microplate Procedure (Sample to WR ratio = 1:8) (3)Troubleshooting (4)Related Thermo Scientific Products (5)Additional Information (5)References (6)IntroductionThe Thermo Scientific Pierce BCA Protein Assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein. This method combines the well-known reduction of Cu+2 to Cu+1 by protein in an alkaline medium (the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+1) using a unique reagent containing bicinchoninic acid.1 The purple-colored reaction product of this assay is formed by the chelation of two molecules of BCA with one cuprous ion. This water-soluble complex exhibits a strong absorbance at 562nm that is nearly linear with increasing protein concentrations over a broad working range (20-2000µg/mL). The BCA method is not a true end-point method; that is, the final color continues to develop. However, following incubation, the rate of continued color development is sufficiently slow to allow large numbers of samples to be assayed together.The macromolecular structure of protein, the number of peptide bonds and the presence of four particular amino acids (cysteine, cystine, tryptophan and tyrosine) are reported to be responsible for color formation with BCA.2 Studies with di-, tri- and tetrapeptides suggest that the extent of color formation caused by more than the mere sum of individual color-producing functional groups.2 Accordingly, protein concentrations generally are determined and reported with reference to standards of a common protein such as bovine serum albumin (BSA). A series of dilutions of known concentration are prepared from the protein and assayed alongside the unknown(s) before the concentration of each unknown is determined based on the standard curve. If precise quantitation of an unknown protein is required, it is advisable to select a proteinstandard that is similar in quality to the unknown; for example, a bovine gamma globulin (BGG) standard (see Related Thermo Scientific Products) may be used when assaying immunoglobulin samples.Two assay procedures are presented. Of these, the Test Tube Procedure requires a larger volume (0.1mL) of protein sample; however, because it uses a sample to working reagent ratio of 1:20 (v/v), the effect of interfering substances is minimized. The Microplate Procedure affords the sample handling ease of a microplate and requires a smaller volume (10-25µL) of protein sample; however, because the sample to working reagent ratio is 1:8 (v/v), it offers less flexibility in overcoming interfering substance concentrations and obtaining low levels of detection.Preparation of Standards and Working Reagent (required for both assay procedures) A.Preparation of Diluted Albumin (BSA) StandardsUse Table 1 as a guide to prepare a set of protein standards. Dilute the contents of one Albumin Standard (BSA) ampule into several clean vials, preferably using the same diluent as the sample(s). Each 1mL ampule of 2mg/mL Albumin Standard is sufficient to prepare a set of diluted standards for either working range suggested in Table 1. There will be sufficient volume for three replications of each diluted standard.Table 1. Preparation of Diluted Albumin (BSA) StandardsVial Volume of Diluent(µL)Volume and Source of BSA(µL)Final BSA Concentration(µg/mL)A 0 300 of Stock 2000B 125 375 of Stock 1500C 325 325 of Stock 1000D 175 175 of vial B dilution 750E 325 325 of vial C dilution 500F 325 325 of vial E dilution 250G 325 325 of vial F dilution 125H 400 100 of vial G dilution 25I 400 0 0 = BlankVial Volume of Diluent(µL)Volume and Source of BSA(µL)Final BSA Concentration(µg/mL)A 700 100 of Stock 250B 400 400 of vial A dilution 125C 450 300 of vial B dilution 50D 400 400 of vial C dilution 25E 400 100 of vial D dilution 5F 400 0 0 = BlankB.Preparation of the BCA Working Reagent (WR)e the following formula to determine the total volume of WR required:(# standards + # unknowns) × (# replicates) × (volume of WR per sample) = total volume WR required Example: for the standard test-tube procedure with 3 unknowns and 2 replicates of each sample:(9 standards + 3 unknowns) × (2 replicates) × (2mL) = 48mL WR requiredNote: 2.0mL of the WR is required for each sample in the test-tube procedure, while only 200 µl of WR reagent is required for each sample in the microplate procedure.2.Prepare WR by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1, Reagent A:B). For the aboveexample, combine 50mL of Reagent A with 1mL of Reagent B.Note: When Reagent B is first added to Reagent A, turbidity is observed that quickly disappears upon mixing to yield a clear, green WR. Prepare sufficient volume of WR based on the number of samples to be assayed. The WR is stable for several days when stored in a closed container at room temperature (RT).Procedure Summary (Test-tube Procedure, Standard Protocol)Test-tube Procedure (Sample to WR ratio = 1:20)1.Pipette 0.1mL of each standard and unknown sample replicate into an appropriately labeled test tube.2.Add 2.0mL of the WR to each tube and mix well.3.Cover and incubate tubes at selected temperature and time:•Standard Protocol: 37°C for 30 minutes (working range = 20-2000µg/mL)•RT Protocol: RT for 2 hours (working range = 20-2000µg/mL)•Enhanced Protocol: 60°C for 30 minutes (working range = 5-250µg/mL)Notes:•Increasing the incubation time or temperature increases the net 562nm absorbance for each test and decreases both the minimum detection level of the reagent and the working range of the protocol.•Use a water bath to heat tubes for either Standard (37°C incubation) or Enhanced (60°C incubation) Protocol. Usinga forced-air incubator can introduce significant error in color development because of uneven heat transfer.4.Cool all tubes to RT.5.With the spectrophotometer set to 562nm, zero the instrument on a cuvette filled only with water. Subsequently, measurethe absorbance of all the samples within 10 minutes.Note: Because the BCA assay does not reach a true end point, color development will continue even after cooling to RT.However, because the rate of color development is low at RT, no significant error will be introduced if the 562nm absorbance measurements of all tubes are made within 10 minutes of each other.6.Subtract the average 562nm absorbance measurement of the Blank standard replicates from the 562nm absorbancemeasurement of all other individual standard and unknown sample replicates.7.Prepare a standard curve by plotting the average Blank-corrected 562nm measurement for each BSA standard vs. itsconcentration in µg/mL. Use the standard curve to determine the protein concentration of each unknown sample. Microplate Procedure (Sample to WR ratio = 1:8)1.Pipette 25µL of each standard or unknown sample replicate into a microplate well (working range = 20-2000µg/mL).Note: If sample size is limited, 10µL of each unknown sample and standard can be used (sample to WR ratio = 1:20).However, the working range of the assay in this case will be limited to 125-2000µg/mL.2.Add 200µL of the WR to each well and mix plate thoroughly on a plate shaker for 30 seconds.3.Cover plate and incubate at 37°C for 30 minutes.4.Cool plate to RT. Measure the absorbance at or near 562nm on a plate reader.Notes:•Wavelengths from 540-590nm have been used successfully with this method.•Because plate readers use a shorter light path length than cuvette spectrophotometers, the Microplate Procedure requires a greater sample to WR ratio to obtain the same sensitivity as the standard Test Tube Procedure. If higher 562nm measurements are desired, increase the incubation time to 2 hours.•Increasing the incubation time or ratio of sample volume to WR increases the net 562nm measurement for each well and lowers both the minimum detection level of the reagent and the working range of the assay. As long as allstandards and unknowns are treated identically, such modifications may be useful.5.Subtract the average 562nm absorbance measurement of the Blank standard replicates from the 562nm measurements ofall other individual standard and unknown sample replicates.6.Prepare a standard curve by plotting the average Blank-corrected 562nm measurement for each BSA standard vs. itsconcentration in µg/mL. Use the standard curve to determine the protein concentration of each unknown sample.Note: If using curve-fitting algorithms associated with a microplate reader, a four-parameter (quadratic) or best-fit curve will provide more accurate results than a purely linear fit. If plotting results by hand, a point-to-point curve is preferable to a linear fit to the standard points.A.Interfering substancesCertain substances are known to interfere with the BCA assay including those with reducing potential, chelating agents, and strong acids or bases. Because they are known to interfere with protein estimation at even minute concentrations, avoid the following substances as components of the sample buffer:Ascorbic Acid EGTA Iron Impure SucroseCatecholamines Impure Glycerol Lipids TryptophanCreatinine Hydrogen Peroxide Melibiose TyrosineCysteine Hydrazides Phenol Red Uric AcidOther substances interfere to a lesser extent with protein estimation using the BCA assay, and these have only minor (tolerable) effects below a certain concentration in the original sample. Maximum compatible concentrations for many substances in the Standard Test Tube Protocol are listed in Table 2 (see last page of Instructions). Substances were compatible at the indicated concentration in the Standard Test Tube Protocol if the error in protein concentration estimation caused by the presence of the substance was less than or equal to 10%. The substances were tested using WR prepared immediately before each experiment. Blank-corrected 562nm absorbance measurements (for a 1000µg/mL BSA standard + substance) were compared to the net 562nm measurements of the same standard prepared in 0.9% saline. Maximum compatible concentrations will be lower In the Microplate Procedure where the sample to WR ratio is 1:8 (v/v). Furthermore, it is possible to have a substance additive affect such that even though a single component is present at a concentration below its listed compatibility, a sample buffer containing a combination of substances could interfere with the assay.B.Strategies for eliminating or minimizing the effects of interfering substancesThe effects of interfering substances in the Pierce BCA Protein Assay may be eliminated or overcome by one of several methods. •Remove the interfering substance by dialysis or gel filtration.•Dilute the sample until the substance no longer interferes. This strategy is effective only if the starting protein concentration is sufficient to remain in the working range of the assay upon dilution.•Precipitate the proteins in the sample with acetone or trichloroacetic acid (TCA). The liquid containing the substance that interfered is discarded and the protein pellet is easily solubilized in ultrapure water or directly in the alkaline BCA WR.4A protocol detailing this procedure is available from our website. Alternatively, Product No. 23215 may be used (seeRelated Pierce Products).•Increase the amount of copper in the WR (prepare WR as 50:2 or 50:3, Reagent A:B), which may eliminate interference by copper-chelating agents.Note: For greatest accuracy, the protein standards must be treated identically to the sample(s).Related Thermo Scientific Products15041 Pierce 96-Well Plates, 100/pkg.15075 Reagent Reservoirs, 200/pkg.15036 Sealing Tape for 96-Well Plates, 100/pkg.23209 Albumin Standard Ampules, 2mg/mL, 10 × 1mL ampules, containing bovine serum albumin (BSA) 23208 Pre-Diluted Protein Assay Standards: Bovine Serum Albumin (BSA) Set, 7 × 3.5mL23212 Bovine Gamma Globulin Standard, 2mg/mL, 10 × 1mL ampules23213 Pre-Diluted Protein Assay Standards, (BGG) Set, 7 × 3.5mL aliquots23235 Pierce Micro BCA Protein Assay Kit, working range of 0.5-20µg/mL23236 Coomassie Plus (Bradford) Assay Kit, working range of 1-1500µg/mL23215 Compat-Able™ Protein Assay Preparation Reagent Set23250Pierce BCA Protein Assay Kit−Reducing Agent CompatibleAdditional InformationA.Please visit our website for additional information including the following items:•Frequently Asked Questions•Tech Tip protocol: Eliminate interfering substances from samples for BCA Protein AssayB.Alternative Total Protein Assay ReagentsIf interference by a reducing substance or metal-chelating substance contained in the sample cannot be overcome, try the Thermo Scientific Coomassie Plus (Bradford) Assay Kit (Product No. 23236), which is less sensitive to such substances.C.Cleaning and Re-using GlasswareExercise care when re-using glassware. All glassware must be cleaned and given a thorough final rinse with ultrapure water.D.Response characteristics for different proteinsEach of the commonly used total protein assay methods exhibits some degree of varying response toward different proteins. These differences relate to amino acid sequence, pI, structure and the presence of certain side chains or prosthetic groups that can dramatically alter the protein’s color response. Most protein assay methods use BSA or immunoglobulin (IgG) as the standard against which the concentration of protein in the sample is determined (Figure 1). However, if great accuracy is required, prepare the standard curve from a pure sample of the target protein.Typical protein-to-protein variation in color response is listed in Table 3. All proteins were tested at 1000µg/mL using the 30-minute/37°C Test Tube Protocol. The average net color response for BSA was normalized to 1.00 and the average net color response of the other proteins is expressed as a ratio to the response of BSA.Figure 1: Typical color response curves for BSA and BGG using the Standard Test Tube Protocol (37°C/30-minute incubation). Table 3. Protein-to-protein variation. Absorbance ratios (562nm) for proteins relative to BSA using Protein Tested Ratio Albumin, bovine serum 1.00 Aldolase, rabbit muscle 0.85 α-Chymotrypsinogen, bovine 1.14 Cytochrome C, horse heart 0.83 Gamma globulin, bovine1.11 IgG, bovine 1.21 IgG, human 1.09 IgG, mouse 1.18 IgG, rabbit 1.12 IgG, sheep1.17 Insulin, bovine pancreas 1.08 Myoglobin, horse heart0.74 Ovalbumin 0.93 Transferrin, human 0.891.02 Standard Deviation 0.15Coefficient of Variation14.7%Cited References1. Smith, P.K., et al. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem . 150:76-85.2. Wiechelman, K., et al. (1988). Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation. Anal Biochem . 175:231-7.3. Kessler, R. and Fanestil, D. (1986). Interference by lipids in the determination of protein using bicinchoninic acid. Anal. Biochem . 159:138-42.4.Brown, R., et al. (1989). Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal. Biochem . 180:136-9.Product ReferencesAdilakshami, T. and Laine, R.O. (2002). Ribosomal protein S25 mRNA partners with MTF-1 and La to provide a p53-mediated mechanism for survival ordeath. J. Biol. Chem. 277:4147-51.Fischer, T., et al. (1999). Clathrin-coated vesicles bearing GAIP possess GTPase-activating protein activity in vitro. Proc. Nat. Acad. Sci. 96:6722-7. Prozialeck, W.C., et al. (2002). Chlamydia trachomatis disrupts N-cadherin-dependent cell-cell junctions and sequester β-catenin in human cervicalepithelial cells. Infection and Immunity 70:2605-13.Roberts, K.P., et al. (2002). A comparative analysis of expression and processing of the rat epididymal fluid and sperm-bound forms of proteins D and E.Biology of Reproduction 67:525-33.Triton ® is a registered trademark of Rohm & Haas Co.Brij ®, Tween ® and Span ® are registered trademarks of ICI Americas. Zwittergent ® is a registered trademark of American Hoechst Corporation.This product (“Product”) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as set forth in the Product documentation, specifications and/or accompanying package inserts (“Documentation”) and to be free from defects in material and workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to anyone other than the original purchaser of the Product (“Buyer”).No other warranties, express or implied, are granted, including without limitation, implied warranties of merchantability, fitness for any particular purpose, or non infringement. Buyer’s exclusive remedy for non-conforming Products during the warranty period is limited to replacement of or refund for the non-conforming Product(s).There is no obligation to replace Products as the result of (i) accident, disaster or event of force majeure, (ii) misuse, fault or negligence of or by Buyer, (iii) use of the Products in a manner for which they were not designed, or (iv) improper storage and handling of the Products.Current product instructions are available at /pierce . For a faxed copy, call 800-874-3723 or contact your local distributor. © 2011 Thermo Fisher Scientific Inc. All rights reserved. Unless otherwise indicated, all trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries. Printed in the USA.Table 2. Compatible substance concentrations in the BCA Protein Assay (see text for details).§* Diluted with ultrapure water.** Detergents were tested using high-purity Thremo Scientific Surfact-Amps Products, which have low peroxide content.-- Dashed-line entry indicates that the material is incompatible with the assay.§ For a more extensive list of substances, download Tech Tip # 68: Protein Assay Compatibility Table from our website. This Tech Tip includes compatible substances for all of our protein assays and enables easy comparisons.。

BCA蛋白定量试剂盒配制及内部操作SOP一、BCA试剂盒配制流程1、标准品配制：配制2mg/ml的BSA标准品，以50ml为例，各成分加入量如下表：原料浓度加入量（mg）厂家货号牛血清白蛋白（BSA）0.2%100北京（二楼生产部提供）YSCW2500氯化钠（NaCl）0.9%450国药集团化学试剂有限公司10019308叠氮化钠0.05%25成都金山化学试剂有限公司/注意：为了确保检测的准确性，应用微量天平精准称量100mg，然后定容至50ml，1ml/管分装，4℃保存。

2、工作液配制由于A液和B液加入比例为50:1，因此配制工作液时A液各成分加入量按照1000ml配制量加入，B液成分按照25ml配制量加入。

原料浓度加入量（g）厂家货号A液（1000ml）二喹啉甲酸（BCA）1％10aladdin B107658无水碳酸钠2％20天津博迪化工股份有限公司/碳酸氢钠0.95%9.5国药集团化学试剂有限公司10018960酒石酸钠0.16% 1.6国药集团化学试剂有限公司30169818氢氧化钠0.4％4国药集团化学试剂有限公司10019762B液（25ml）硫酸铜4％1国药集团化学试剂有限公司81005261备注：A液各成分混合之后，调节PH至11.25。

二、BCA法蛋白定量操作SOP1、标准品稀释按照下表制备一组蛋白质标准品。

将一安瓿的牛血清白蛋白标准品（BSA）稀释到几个干净的小瓶中，最好使用与待测样品相同的缓冲液。

每一个1mL安瓿的2mg/mL牛血清白蛋白标准品足够用于制备下表中所列出的任意一组稀释范围的标准品；每个稀释浓度的标准品的体积足够用于3次重复检测。

用于标准方案的稀释方法（检测范围=20-2,000ug/mL）如下：标准液编号稀释液体积（ul）标准品体积（ul）标准液终浓度（ug/ml）A03002000B125375ul的A1500C325325ul的A1000D175175ul的B750E325325ul的C500F325325ul的E250G325325ul的F125H400100ul的G25I40000用于试管增强方案的稀释方法如下（检测范围=5–250ug/mL）：标准液编号稀释液体积（ul）标准品体积（ul）标准液终浓度（ug/ml）A700100250B400400ul的A125C450300ul的B50D400400ul的C25E400100ul的D5F400002.样品稀释：根据需要，将样品用用PBS或者与待测样品缓冲液相同的溶液稀释至线性范围内（所用稀释液与标准品稀释液应保持一致）。

BCA测蛋白的具体操作步骤材料准备：1.BCA试剂盒：包括BCA试剂A、BCA试剂B和BCA标准品。

2.待测蛋白样本：可以是纯化的蛋白质溶液或细胞裂解液。

3.96孔酶标板：透明底板。

4.微量移液器和相应的移液器头垫片。

5.离心机。

操作步骤：1.准备BCA标准曲线：a.将BCA标准品1-5按照不同的浓度配制好。

可以根据需要使用不同浓度的标准品，但至少需要配制3个浓度的标准品。

b.分别取BCA标准品和蒸馏水作为白板，每组加入200μL的BCA试剂A和200μL的BCA试剂B，混匀。

c. 在37°C恒温下孵育30分钟后，读取吸光度值。

一般可在562 nm波长处进行测量。

2.准备待测样品：a. 根据需要，将蛋白样品稀释到适当的浓度，通常需要在0.1-1.0 mg/mL之间。

b.取等体积的蛋白质溶液和蒸馏水作为空白对照。

例如，取200μL 的蛋白质溶液和200μL的蒸馏水。

3.进行BCA测定：a.取一个96孔酶标板，在需要测定的样品孔中分别加入25μL的蛋白质样品和25μL的蒸馏水作为空白对照。

b.加入200μL的BCA试剂A和200μL的BCA试剂B到每个孔中，混匀。

确保试剂能与蛋白样品充分接触。

c.在37°C恒温下孵育30分钟。

在孵育期间，可以同时进行标准品的测定。

d.孵育结束后，使用微量移液器将样品转移到一个透明底的96孔读板中。

如果有大量沉淀物，可以在离心后将上清转移到读板中。

e. 在562 nm波长处读取吸光度值。

4.绘制标准曲线和计算样品浓度：a.使用BCA标准曲线的吸光度值计算每个标准品的平均吸光度。

b.将标准品的平均吸光度与浓度绘制成标准曲线。

c.使用样品的吸光度值和标准曲线，计算样品中蛋白质的浓度。

5.数据分析：a.根据样品的吸光度值和标准曲线，计算出样品中蛋白质的浓度。

b.可以根据需要将浓度值进行转换，比如将浓度值换算为蛋白质的摩尔浓度。

注意事项：1.所有操作都需要在洁净的实验台上进行，以避免外源性污染对结果的影响。

INSTRUCTIONSWarranty: Pierce products are warranted to meet stated product specifications and to conform to label descriptions when used and stored properly. Unless otherwise stated, this warranty is limited to one year from date of sale for products used, handled and stored according to Pierce instructions. Pierce’s sole liability for the product is limited toNumberDescription23225BCA™ Protein Assay Kit , sufficient reagents for 500 test tube or 5,000 microplate assays 23227BCA™ Protein Assay Kit , sufficient reagents for 250 test tube or 2,500 microplate assays Kit Contents:BCA™ Reagent A , 1,000 ml (in Product No. 23225) or 500 ml (in Product No. 23227), containing sodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartrate in 0.1 M sodium hydroxideBCA™ Reagent B , 25 ml, containing 4% cupric sulfateAlbumin Standard Ampules, 2 mg/ml , 10 x 1 ml ampules, containing bovine serum albumin (BSA)at 2.0 mg/ml in 0.9% saline and 0.05% sodium azideStorage: Upon receipt store at room temperature. Product shipped at ambient temperature.Note: If either Reagent A or Reagent B precipitates upon shipping in cold weather or during long-term storage, dissolve precipitates by gently warming and stirring solution. Discard any kit reagent that shows discoloration or evidence of microbial contamination.Table of ContentsIntroduction..................................................................................................................................................................................1Preparation of Standards and Working Reagent (required for both assay procedures)................................................................2Test Tube Procedure (Sample to WR ratio = 1:20).....................................................................................................................3Microplate Procedure (Sample to WR ratio = 1:8)......................................................................................................................3Troubleshooting...........................................................................................................................................................................4Related Pierce Products...............................................................................................................................................................5Additional Information................................................................................................................................................................5Cited References..........................................................................................................................................................................6Product References. (6)IntroductionThe BCA™ Protein Assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein. This method combines the well-known reduction of Cu +2 to Cu +1 by protein in an alkaline medium (the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu +1) using a unique reagent containing bicinchoninic acid.1 The purple-colored reaction product of this assay is formed by the chelation of two molecules of BCA with one cuprous ion. This water-soluble complex exhibits a strong absorbance at 562 nm that is nearly linear with increasing protein concentrations over a broad working range (20-2,000 µg/ml). The BCA™method is not a true end-point method; that is, the final color continues to develop. However, following incubation, the rate of continued color development is sufficiently slow to allow large numbers of samples to be assayed together.The macromolecular structure of protein, the number of peptide bonds and the presence of four particular amino acids (cysteine, cystine, tryptophan and tyrosine) are reported to be responsible for color formation with BCA.2 Studies with di-,tri- and tetrapeptides suggest that the extent of color formation caused by more than the mere sum of individual color-producing functional groups.2 Accordingly, protein concentrations generally are determined and reported with reference to standards of a common protein such as bovine serum albumin (BSA). A series of dilutions of known concentration are3747 N. Meridian Road P.O. Box 117Rockford, IL 61105BCA™ Protein Assay Kitprepared from the protein and assayed alongside the unknown(s) before the concentration of each unknown is determined based on the standard curve. If precise quantitation of an unknown protein is required, it is advisable to select a protein standard that is similar in quality to the unknown; for example, a bovine gamma globulin (BGG) standard (see Related Pierce Products) may be used when assaying immunoglobulin samples.Two assay procedures are presented. Of these, the Test Tube Procedure requires a larger volume (0.1 ml) of protein sample; however, because it uses a sample to working reagent ratio of 1:20 (v/v), the effect of interfering substances is minimized. The Microplate Procedure affords the sample handling ease of a microplate and requires a smaller volume (10-25 µl) of protein sample; however, because the sample to working reagent ratio is 1:8 (v/v), it offers less flexibility in overcoming interfering substance concentrations and obtaining low levels of detection.Preparation of Standards and Working Reagent (required for both assay procedures) A.Preparation of Diluted Albumin (BSA) StandardsUse Table 1 as a guide to prepare a set of protein standards. Dilute the contents of one Albumin Standard (BSA) ampule into several clean vials, preferably using the same diluent as the sample(s). Each 1 ml ampule of 2.0 mg/ml Albumin Standard is sufficient to prepare a set of diluted standards for either working range suggested in Table 1. There will be sufficient volume for three replications of each diluted standard.Table 1. Preparation of Diluted Albumin (BSA) StandardsDilution Scheme for Standard Test Tube Protocol and Microplate Procedure (Working Range = 20–2,000 µg/ml) Vial Volume of Diluent Volume and Source of BSA Final BSA ConcentrationA0300 µl of Stock2,000 µg/mlB125 µl375 µl of Stock1,500 µg/mlC325 µl325 µl of Stock1,000 µg/mlD175 µl175 µl of vial B dilution750 µg/mlE325 µl325 µl of vial C dilution500 µg/mlF325 µl325 µl of vial E dilution250 µg/mlG325 µl325 µl of vial F dilution125 µg/mlH400 µl100 µl of vial G dilution25 µg/mlI400 µl00 µg/ml = Blank Dilution Scheme for Enhanced Test Tube Protocol (Working Range = 5–250 µg/ml)Vial Volume of Diluent Volume and Source of BSA Final BSA ConcentrationA700 µl100 µl of Stock250 µg/mlB400 µl400 µl of vial A dilution125 µg/mlC450 µl300 µl of vial B dilution50 µg/mlD400 µl400 µl of vial C dilution25 µg/mlE400 µl100 µl of vial D dilution 5 µg/mlF400 µl00 µg/ml = BlankB.Preparation of the BCA™ Working Reagent (WR)e the following formula to determine the total volume of WR required:(# standards + # unknowns) x (# replicates) x (volume of WR per sample) = total volume WR required Example: for the Standard Test Tube Protocol with 3 unknowns and 2 replicates of each sample:(9 standards + 3 unknowns) x (2 replicates) x (2 ml) = 48 ml WR requiredNote: 2.0 ml of the WR is required for each sample in the Test Tube Procedure, while only 200 µl of WR reagent is required for each sample in the Microplate Procedure.2.Prepare WR by mixing 50 parts of BCA™ Reagent A with 1 part of BCA™ Reagent B (50:1, Reagent A:B). For theabove example, combine 50 ml of Reagent A with 1 ml of Reagent B.Note: When Reagent B is first added to Reagent A, a turbidity is observed that quickly disappears upon mixing to yield a clear, green WR. Prepare sufficient volume of WR based on the number of samples to be assayed. The WR is stable for several days when stored in a closed container at room temperature (RT).Procedure Summary (Test Tube Procedure, Standard Protocol)Test Tube Procedure (Sample to WR ratio = 1:20)1.Pipette 0.1 ml of each standard and unknown sample replicate into an appropriately labeled test tube.2.Add 2.0 ml of the WR to each tube and mix well.3.Cover and incubate tubes at selected temperature and time:•Standard Protocol:37°C for 30 minutes (working range = 20-2,000 µg/ml)•RT Protocol:RT for 2 hours (working range = 20-2,000 µg/ml)•Enhanced Protocol:60°C for 30 minutes (working range = 5-250 µg/ml)Notes:•Increasing the incubation time or temperature increases the net 562 nm absorbance for each test and decreases both the minimum detection level of the reagent and the working range of the protocol.•Use a water bath to heat tubes for either Standard (37°C incubation) or Enhanced (60°C incubation) Protocol. Usinga forced-air incubator can introduce significant error in color development because of uneven heat transfer.4.Cool all tubes to RT.5.With the spectrophotometer set to 562 nm, zero the instrument on a cuvette filled only with water. Subsequently,measure the absorbance of all the samples within 10 minutes.Note: Because the BCA™ Assay does not reach a true end point, color development will continue even after cooling to RT. However, because the rate of color development is low at RT, no significant error will be introduced if the 562 nm absorbance measurements of all tubes are made within 10 minutes of each other.6.Subtract the average 562 nm absorbance measurement of the Blank standard replicates from the 562 nm absorbancemeasurement of all other individual standard and unknown sample replicates.7.Prepare a standard curve by plotting the average Blank-corrected 562 nm measurement for each BSA standard vs. itsconcentration in µg/ml. Use the standard curve to determine the protein concentration of each unknown sample. Microplate Procedure (Sample to WR ratio = 1:8)1.Pipette 25 µl of each standard or unknown sample replicate into a microplate well (working range = 20-2,000 µg/ml).Note: If sample size is limited, 10 µl of each unknown sample and standard can be used (sample to WR ratio = 1:20).However, the working range of the assay in this case will be limited to 125-2,000 µg/ml.2.Add 200 µl of the WR to each well and mix plate thoroughly on a plate shaker for 30 seconds.3.Cover plate and incubate at 37°C for 30 minutes.4.Cool plate to RT.5.Measure the absorbance at or near 562 nm on a plate reader.Notes:•Wavelengths from 540-590 nm have been used successfully with this method.•Because plate readers use a shorter light path length than cuvette spectrophotometers, the Microplate Procedure requires a greater sample to WR ratio to obtain the same sensitivity as the standard Test Tube Procedure. If higher 562 nm measurements are desired, increase the incubation time to 2 hours.•Increasing the incubation time or ratio of sample volume to WR increases the net 562 nm measurement for each well and lowers both the minimum detection level of the reagent and the working range of the assay. As long as all standards and unknowns are treated identically, such modifications may be useful.6. Subtract the average 562 nm absorbance measurement of the Blank standard replicates from the 562 nm measurementsof all other individual standard and unknown sample replicates.7. Prepare a standard curve by plotting the average Blank-corrected 562 nm measurement for each BSA standard vs. itsconcentration in µg/ml. Use the standard curve to determine the protein concentration of each unknown sample.Note: If using curve-fitting algorithms associated with a microplate reader, a four-parameter (quadratic) or best-fit curve will provide more accurate results than a purely linear fit. If plotting results by hand, a point-to-point curve is preferable to a linear fit to the standard points.TroubleshootingProblemPossible CauseSolutionNo color in any tubesSample contains a copper chelating agentDialyze, desalt, or dilute sampleIncrease copper concentration in working reagent (e.g., use 50:2, Reagent A:B)Remove interfering substances from sample using Product No. 23215Strong acid or alkaline buffer, alters working reagent pHDialyze, desalt, or dilute sample Blank absorbance is OK, but standards and samples show less color than expectedColor measured at the wrong wavelengthMeasure the absorbance at 562 nm Protein concentration is too high Dilute sampleColor of samples appears darker than expectedSample contains lipids or lipoproteinsAdd 2% SDS to the sample to eliminate interference from lipids 3Remove interfering substances from sample using Product No. 23215Buffer contains a reducing agent Buffer contains a thiolAll tubes (including blank) are dark purpleBuffer contains biogenic amines (catecholamines)Dialyze or dilute sampleRemove interfering substances from sample using Product No. 23215Need to measure color at a different wavelengthSpectrophotometer or plate reader does not have 562 nm filterColor may be measure at any wavelength between 540 nm and 590 nm, although the slope of standard curve and overall assay sensitivity will be reducedA. Interfering substancesCertain substances are known to interfere with the BCA™ Assay including those with reducing potential, chelating agents,and strong acids or bases. Because they are known to interfere with protein estimation at even minute concentrations, avoid the following substances as components of the sample buffer:Ascorbic Acid EGTAIron Impure Sucrose Catecholamines Impure Glycerol Lipids Tryptophan Creatinine Hydrogen Peroxide Melibiose Tyrosine CysteineHydrazidesPhenol RedUric AcidOther substances interfere to a lesser extent with protein estimation using the BCA™ Assay, and these have only minor (tolerable) effects below a certain concentration in the original sample. Maximum compatible concentrations for many substances in the Standard Test Tube Protocol are listed in Table 2 (see last page of Instructions). Substances werecompatible at the indicated concentration in the Standard Test Tube Protocol if the error in protein concentration estimation caused by the presence of the substance in the sample was less than or equal to 10%. The substances were tested using WR prepared immediately before each experiment. Blank-corrected 562 nm absorbance measurements (for a 1,000 µg/ml BSA standard + substance) were compared to the net 562 nm measurements of the same standard prepared in 0.9% saline. In the Microplate Procedure, where the sample to WR ratio is 1:8 (v/v), maximum compatible concentrations will be lower.B.Strategies for eliminating or minimizing the effects of interfering substancesThe effects of interfering substances in the BCA™ Protein Assay may be eliminated or overcome by one of several methods.•Remove the interfering substance by dialysis or gel filtration.•Dilute the sample until the substance no longer interferes. This strategy is effective only if the starting protein concentration is sufficient to remain in the working range of the assay upon dilution.•Precipitate the proteins in the sample with acetone or trichloroacetic acid (TCA). The liquid containing the substance that interfered is discarded and the protein pellet is easily solubilized in ultrapure water or directly in the alkaline BCA™WR.4 A protocol for performing this on samples to be assayed with BCA™ Protein Assay Reagent is available at the Pierce web site. Alternatively, Product No. 23215 may be used (see Related Pierce Products).•Increase the amount of copper in the WR (prepare WR as 50:2 or 50:3, Reagent A:B), which may eliminate interference by copper chelating agents.Note: For greatest accuracy, the protein standards must be treated identically to the sample(s).Related Pierce Products23209Albumin Standard Ampules, 2 mg/ml, 10 x 1 ml ampules, containing bovine serum albumin (BSA) at 2.0 mg/ml in 0.9% saline and 0.05% sodium azide23208Pre-Diluted Protein Assay Standards: Bovine Serum Albumin (BSA) Set, 7 x 3.5 ml aliquots in the range of 125-2,000 µg/ml23212Bovine Gamma Globulin Standard, 2 mg/ml, 10 x 1 ml ampules23213Pre-Diluted Protein Assay Standards, Bovine Gamma Globulin Fraction II (BGG) Set, 7 x 3.5 ml aliquots in the range of 125-2,000 µg/ml23221BCA™ Reagent A, 1,000 ml23223BCA™ Reagent A, 250 ml23224BCA™ Reagent B, 25 ml23235Micro BCA TM Protein Assay Kit, working range of 0.5-20 µg/ml23236Coomassie Plus™ Protein Assay Kit, working range of 1-1,500 µg/ml23215Compat-Able TM Protein Assay Preparation Reagent Set, sufficient reagents to pre-treat 500 samples to remove interfering substances before total protein quantitationAdditional InformationA.Please visit the Pierce web site for additional information on this product including the following items:•Frequently Asked Questions•Tech Tip protocol: Eliminate interfering substances from samples for BCA™ Protein Assay•Tech Tip protocol: Shorten BCA™ Protein Assay incubation using a microwave ovenB.Response characteristics for different proteinsEach of the commonly used total protein assay methods exhibits some degree of varying response toward different proteins. These differences relate to amino acid sequence, pI, structure and the presence of certain side chains or prosthetic groups that can dramatically alter the protein’s color response. Most protein assay methods utilize BSA or immunoglobulin (IgG) as the standard against which the concentration of protein in the sample is determined (Figure 1). However, if great accuracy is required, the standard curve should be prepared from a pure sample of the target protein to be measured.Table 3 shows typical BCA™ Protein Assay protein-to-protein variation in color response. All proteins were tested at a concentration of 1,000 µg/ml using the 30-minute/37°C Test Tube Protocol. The average net color response for BSA was normalized to 1.00 and the average net color response of the other proteins is expressed as a ratio to the response of BSA.Table 3. Protein-to-Protein Variation. Absorbance ratios (562 nm) for proteins relative to BSA using the Standard Test Tube Protocol.Ratio = (Avg “test” net Abs.) / (avg. BSA net Abs.)Protein Tested Ratio Albumin, bovine serum 1.00Aldolase, rabbit muscle 0.85α-Chymotrypsinogen, bovine 1.14Cytochrome C, horse heart 0.83Gamma globulin, bovine1.11IgG, bovine 1.21IgG, human 1.09IgG, mouse 1.18IgG, rabbit 1.12IgG, sheep1.17Insulin, bovine pancreas 1.08Myoglobin, horse heart0.74Ovalbumin 0.93Transferrin, human0.89Average ratio1.02Figure 1: Typical color response curves for BSA and BGG using the Standard Test Tube Protocol (37°C/30-minute incubation).Standard Deviation 0.15Coefficient of Variation14.7%C. Alternative Total Protein Assay ReagentsIf interference by a reducing substance or metal-chelating substance contained in the sample cannot be overcome, try the Coomassie Plus™ Protein Assay Kit (Product No. 23236), which is less sensitive to such substances.D. Cleaning and Re-using GlasswareExercise care when re-using glassware. All glassware must be cleaned and given a thorough final rinse with ultrapure water.Cited References1. Smith, P.K., et al. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem . 150:76-85.2. Wiechelman, K., Braun, R. and Fitzpatrick, J. (1988). Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation. Anal Biochem . 175:231-7.3. Kessler, R. and Fanestil, D. (1986). Interference by lipids in the determination of protein using bicinchoninic acid. Anal. Biochem . 159:138-42.4.Brown, R., Jarvis, K. and Hyland, K. (1989). Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal. Biochem .180:136-9.Product ReferencesAdilakshami, T. and Laine, R.O. (2002). Ribosomal protein S25 mRNA partners with MTF-1 and La to provide a p53-mediated mechanism for survival ordeath. J. Biol. Chem. 277:4147-51.Fischer, T., et al. (1999). Clathrin-coated vesicles bearing GAIP possess GTPase-activating protein activity in vitro. Proc. Nat. Acad. Sci. 96:6722-7.Prozialeck, W.C., et al. (2002). Chlamydia trachomatis disrupts N-cadherin-dependent cell-cell junctions and sequester β-catenin in human cervicalepithelial cells. Infection and Immunity 70:2605-13.Roberts, K.P., Ensrud, K.M. and Hamilton, D.W. (2002). A comparative analysis of expression and processing of the rat epididymal fluid and sperm-boundforms of proteins D and E. Biology of Reproduction 67:525-33.Triton ® is a registered trademark of Rohm & Haas Co.Brij ®, Tween ® and Span ® are registered trademarks of ICI Americas.Zwittergent ® is a registered trademark of American Hoechst Corporation.The BCA™ Protein Assay is protected by U.S. Patent # 4,839,295©Pierce Biotechnology, Inc., 10/2003. Printed in the USA.Table 2. Compatible Substance Concentrations in the BCA™ Protein Assay (see text for details).Substance CompatibleConcentration Salts/BuffersACES, pH 7.825 mM Ammonium sulfate 1.5 M Asparagine 1 mMBicine, pH 8.420 mMBis-Tris, pH 6.533 mMBorate (50 mM), pH 8.5 (# 28384)undilutedB-PER® Reagent (#78248)undiluted Calcium chloride in TBS, pH 7.210 mMNa-Carbonate/Na-Bicarbonate (0.2 M),pH 9.4 (#28382)undilutedCesium bicarbonate100 mM CHES, pH 9.0100 mMNa-Citrate (0.6 M), Na-Carbonate (0.1M), pH 9.0 (#28388)1:8 dilution*Na-Citrate (0.6 M), MOPS (0.1 M), pH 7.5(#28386)1:8 dilution*Cobalt chloride in TBS, pH 7.20.8 mM EPPS, pH 8.0100 mMFerric chloride in TBS, pH 7.210 mM Glycine•HCl, pH 2.8100 mM Guanidine•HCl 4 MHEPES, pH 7.5100 mM Imidazole, pH 7.050 mMMES, pH 6.1100 mMMES (0.1 M), NaCl (0.9%), pH 4.7 (#28390)undiluted MOPS, pH 7.2100 mM Modified Dulbecco’s PBS, pH 7.4 (#28374)undilutedNickel chloride in TBS, pH 7.210 mMPBS; Phosphate (0.1 M), NaCl (0.15 M),pH 7.2 (#28372)undiluted PIPES, pH 6.8100 mMRIPA lysis buffer; 50 mM Tris, 150 mM NaCl,0.5% DOC, 1% NP-40, 0.1% SDS, pH 8.0undiluted Sodium acetate, pH 4.8200 mM Sodium azide0.2%Sodium bicarbonate100 mM Sodium chloride 1 MSodium citrate, pH 4.8 or pH 6.4200 mM Sodium phosphate100 mM Tricine, pH 8.025 mM Triethanolamine, pH 7.825 mMTris250 mMTBS; Tris (25 mM), NaCl (0.15 M), pH 7.6(#28376)undilutedTris (25 mM), Glycine (192 mM), pH 8.0(#28380)1:3 dilution*Tris (25 mM), Glycine (192 mM), SDS(0.1%), pH 8.3 (#28378)undilutedZinc chloride in TBS, pH 7.210 mM Substance CompatibleConcentration Detergents**Brij®-35 5.0%Brij®-56, Brij®-58 1.0%CHAPS, CHAPSO 5.0% Deoxycholic acid 5.0%Octyl β-glucoside 5.0%Nonidet P-40 (NP-40) 5.0%Octyl β-thioglucopyranoside 5.0%SDS 5.0%Span® 20 1.0%Triton® X-100 5.0%Triton® X-114, X-305, X-405 1.0%Tween®-20, Tween®-60, Tween®-80 5.0% Zwittergent® 3-14 1.0%Chelating agentsEDTA10 mMEGTA--------Sodium citrate200 mM Reducing & Thiol-Containing AgentsN-acetylglucosamine in PBS, pH 7.210 mM Ascorbic acid--------Cysteine--------Dithioerythritol (DTE) 1 mM Dithiothreitol (DTT) 1 mMGlucose10 mM Melibiose--------2-Mercaptoethanol0.01% Potassium thiocyanate 3.0 M Thimerosal0.01%Misc. Reagents & SolventsAcetone10% Acetonitrile10%Aprotinin10 mg/LDMF, DMSO10%DMSO10%Ethanol10%Glycerol (Fresh)10% Hydrazides--------Hydrides (Na2BH4 or NaCNBH3)--------Hydrochloric Acid100 mM Leupeptin10 mg/L Methanol10%Phenol Red--------PMSF 1 mMSodium Hydroxide100 mM Sucrose40%TLCK0.1 mg/LTPCK0.1 mg/LUrea 3 Mo-Vanadate (sodium salt), in PBS, pH 7.2 1 mM* Diluted with ultrapure water; ** Detergents were tested using Pierce high-purity Surfact-Amps™ Products, which have low peroxide content; -- Dashed-line entry indicates that the material is incompatible with the assay.。

INSTRUCTIONSPierce® BCA Protein Assay Kit23225 Pierce BCA Protein Assay Kit, sufficient reagents for 500 test-tube or 5000 microplate assays 23227 Pierce BCA Protein Assay Kit, sufficient reagents for 250 test-tube or 2500 microplate assays Kit Contents:BCA Reagent A, 1000mL (in Product No. 23225) or 500mL (in Product No. 23227), containingsodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartrate in 0.1M sodiumhydroxideBCA Reagent B, 25mL, containing 4% cupric sulfateAlbumin Standard Ampules, 2mg/mL, 10 × 1mL ampules, containing bovine serum albumin (BSA)at 2mg/mL in 0.9% saline and 0.05% sodium azideStorage: Upon receipt store at room temperature. Product shipped at ambient temperature.Note: If either Reagent A or Reagent B precipitates upon shipping in cold weather or during long-termstorage, dissolve precipitates by gently warming and stirring solution. Discard any kit reagent thatshows discoloration or evidence of microbial contamination.Table of ContentsIntroduction (1)Preparation of Standards and Working Reagent (required for both assay procedures) (2)Test Tube Procedure (Sample to WR ratio = 1:20) (3)Microplate Procedure (Sample to WR ratio = 1:8) (3)Troubleshooting (4)Related Thermo Scientific Products (5)Additional Information (5)References (6)IntroductionThe Thermo Scientific Pierce BCA Protein Assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein. This method combines the well-known reduction of Cu+2 to Cu+1 by protein in an alkaline medium (the biuret reaction) with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+1) using a unique reagent containing bicinchoninic acid.1 The purple-colored reaction product of this assay is formed by the chelation of two molecules of BCA with one cuprous ion. This water-soluble complex exhibits a strong absorbance at 562nm that is nearly linear with increasing protein concentrations over a broad working range (20-2000µg/mL). The BCA method is not a true end-point method; that is, the final color continues to develop. However, following incubation, the rate of continued color development is sufficiently slow to allow large numbers of samples to be assayed together.The macromolecular structure of protein, the number of peptide bonds and the presence of four particular amino acids (cysteine, cystine, tryptophan and tyrosine) are reported to be responsible for color formation with BCA.2 Studies with di-, tri- and tetrapeptides suggest that the extent of color formation caused by more than the mere sum of individual color-producing functional groups.2 Accordingly, protein concentrations generally are determined and reported with reference to standards of a common protein such as bovine serum albumin (BSA). A series of dilutions of known concentration are prepared from the protein and assayed alongside the unknown(s) before the concentration of each unknown is determined based on the standard curve. If precise quantitation of an unknown protein is required, it is advisable to select a proteinstandard that is similar in quality to the unknown; for example, a bovine gamma globulin (BGG) standard (see Related Thermo Scientific Products) may be used when assaying immunoglobulin samples.Two assay procedures are presented. Of these, the Test Tube Procedure requires a larger volume (0.1mL) of protein sample; however, because it uses a sample to working reagent ratio of 1:20 (v/v), the effect of interfering substances is minimized. The Microplate Procedure affords the sample handling ease of a microplate and requires a smaller volume (10-25µL) of protein sample; however, because the sample to working reagent ratio is 1:8 (v/v), it offers less flexibility in overcoming interfering substance concentrations and obtaining low levels of detection.Preparation of Standards and Working Reagent (required for both assay procedures) A.Preparation of Diluted Albumin (BSA) StandardsUse Table 1 as a guide to prepare a set of protein standards. Dilute the contents of one Albumin Standard (BSA) ampule into several clean vials, preferably using the same diluent as the sample(s). Each 1mL ampule of 2mg/mL Albumin Standard is sufficient to prepare a set of diluted standards for either working range suggested in Table 1. There will be sufficient volume for three replications of each diluted standard.Table 1. Preparation of Diluted Albumin (BSA) StandardsVial Volume of Diluent(µL)Volume and Source of BSA(µL)Final BSA Concentration(µg/mL)A 0 300 of Stock 2000B 125 375 of Stock 1500C 325 325 of Stock 1000D 175 175 of vial B dilution 750E 325 325 of vial C dilution 500F 325 325 of vial E dilution 250G 325 325 of vial F dilution 125H 400 100 of vial G dilution 25I 400 0 0 = BlankVial Volume of Diluent(µL)Volume and Source of BSA(µL)Final BSA Concentration(µg/mL)A 700 100 of Stock 250B 400 400 of vial A dilution 125C 450 300 of vial B dilution 50D 400 400 of vial C dilution 25E 400 100 of vial D dilution 5F 400 0 0 = BlankB.Preparation of the BCA Working Reagent (WR)e the following formula to determine the total volume of WR required:(# standards + # unknowns) × (# replicates) × (volume of WR per sample) = total volume WR required Example: for the standard test-tube procedure with 3 unknowns and 2 replicates of each sample:(9 standards + 3 unknowns) × (2 replicates) × (2mL) = 48mL WR requiredNote: 2.0mL of the WR is required for each sample in the test-tube procedure, while only 200 µl of WR reagent is required for each sample in the microplate procedure.2.Prepare WR by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1, Reagent A:B). For the aboveexample, combine 50mL of Reagent A with 1mL of Reagent B.Note: When Reagent B is first added to Reagent A, turbidity is observed that quickly disappears upon mixing to yield a clear, green WR. Prepare sufficient volume of WR based on the number of samples to be assayed. The WR is stable for several days when stored in a closed container at room temperature (RT).Procedure Summary (Test-tube Procedure, Standard Protocol)Test-tube Procedure (Sample to WR ratio = 1:20)1.Pipette 0.1mL of each standard and unknown sample replicate into an appropriately labeled test tube.2.Add 2.0mL of the WR to each tube and mix well.3.Cover and incubate tubes at selected temperature and time:•Standard Protocol: 37°C for 30 minutes (working range = 20-2000µg/mL)•RT Protocol: RT for 2 hours (working range = 20-2000µg/mL)•Enhanced Protocol: 60°C for 30 minutes (working range = 5-250µg/mL)Notes:•Increasing the incubation time or temperature increases the net 562nm absorbance for each test and decreases both the minimum detection level of the reagent and the working range of the protocol.•Use a water bath to heat tubes for either Standard (37°C incubation) or Enhanced (60°C incubation) Protocol. Usinga forced-air incubator can introduce significant error in color development because of uneven heat transfer.4.Cool all tubes to RT.5.With the spectrophotometer set to 562nm, zero the instrument on a cuvette filled only with water. Subsequently, measurethe absorbance of all the samples within 10 minutes.Note: Because the BCA assay does not reach a true end point, color development will continue even after cooling to RT.However, because the rate of color development is low at RT, no significant error will be introduced if the 562nm absorbance measurements of all tubes are made within 10 minutes of each other.6.Subtract the average 562nm absorbance measurement of the Blank standard replicates from the 562nm absorbancemeasurement of all other individual standard and unknown sample replicates.7.Prepare a standard curve by plotting the average Blank-corrected 562nm measurement for each BSA standard vs. itsconcentration in µg/mL. Use the standard curve to determine the protein concentration of each unknown sample. Microplate Procedure (Sample to WR ratio = 1:8)1.Pipette 25µL of each standard or unknown sample replicate into a microplate well (working range = 20-2000µg/mL).Note: If sample size is limited, 10µL of each unknown sample and standard can be used (sample to WR ratio = 1:20).However, the working range of the assay in this case will be limited to 125-2000µg/mL.2.Add 200µL of the WR to each well and mix plate thoroughly on a plate shaker for 30 seconds.3.Cover plate and incubate at 37°C for 30 minutes.4.Cool plate to RT. Measure the absorbance at or near 562nm on a plate reader.Notes:•Wavelengths from 540-590nm have been used successfully with this method.•Because plate readers use a shorter light path length than cuvette spectrophotometers, the Microplate Procedure requires a greater sample to WR ratio to obtain the same sensitivity as the standard Test Tube Procedure. If higher 562nm measurements are desired, increase the incubation time to 2 hours.•Increasing the incubation time or ratio of sample volume to WR increases the net 562nm measurement for each well and lowers both the minimum detection level of the reagent and the working range of the assay. As long as allstandards and unknowns are treated identically, such modifications may be useful.5.Subtract the average 562nm absorbance measurement of the Blank standard replicates from the 562nm measurements ofall other individual standard and unknown sample replicates.6.Prepare a standard curve by plotting the average Blank-corrected 562nm measurement for each BSA standard vs. itsconcentration in µg/mL. Use the standard curve to determine the protein concentration of each unknown sample.Note: If using curve-fitting algorithms associated with a microplate reader, a four-parameter (quadratic) or best-fit curve will provide more accurate results than a purely linear fit. If plotting results by hand, a point-to-point curve is preferable to a linear fit to the standard points.A.Interfering substancesCertain substances are known to interfere with the BCA assay including those with reducing potential, chelating agents, and strong acids or bases. Because they are known to interfere with protein estimation at even minute concentrations, avoid the following substances as components of the sample buffer:Ascorbic Acid EGTA Iron Impure SucroseCatecholamines Impure Glycerol Lipids TryptophanCreatinine Hydrogen Peroxide Melibiose TyrosineCysteine Hydrazides Phenol Red Uric AcidOther substances interfere to a lesser extent with protein estimation using the BCA assay, and these have only minor (tolerable) effects below a certain concentration in the original sample. Maximum compatible concentrations for many substances in the Standard Test Tube Protocol are listed in Table 2 (see last page of Instructions). Substances were compatible at the indicated concentration in the Standard Test Tube Protocol if the error in protein concentration estimation caused by the presence of the substance was less than or equal to 10%. The substances were tested using WR prepared immediately before each experiment. Blank-corrected 562nm absorbance measurements (for a 1000µg/mL BSA standard + substance) were compared to the net 562nm measurements of the same standard prepared in 0.9% saline. Maximum compatible concentrations will be lower In the Microplate Procedure where the sample to WR ratio is 1:8 (v/v). Furthermore, it is possible to have a substance additive affect such that even though a single component is present at a concentration below its listed compatibility, a sample buffer containing a combination of substances could interfere with the assay.B.Strategies for eliminating or minimizing the effects of interfering substancesThe effects of interfering substances in the Pierce BCA Protein Assay may be eliminated or overcome by one of several methods. •Remove the interfering substance by dialysis or gel filtration.•Dilute the sample until the substance no longer interferes. This strategy is effective only if the starting protein concentration is sufficient to remain in the working range of the assay upon dilution.•Precipitate the proteins in the sample with acetone or trichloroacetic acid (TCA). The liquid containing the substance that interfered is discarded and the protein pellet is easily solubilized in ultrapure water or directly in the alkaline BCA WR.4A protocol detailing this procedure is available from our website. Alternatively, Product No. 23215 may be used (seeRelated Pierce Products).•Increase the amount of copper in the WR (prepare WR as 50:2 or 50:3, Reagent A:B), which may eliminate interference by copper-chelating agents.Note: For greatest accuracy, the protein standards must be treated identically to the sample(s).Related Thermo Scientific Products15041 Pierce 96-Well Plates, 100/pkg.15075 Reagent Reservoirs, 200/pkg.15036 Sealing Tape for 96-Well Plates, 100/pkg.23209 Albumin Standard Ampules, 2mg/mL, 10 × 1mL ampules, containing bovine serum albumin (BSA) 23208 Pre-Diluted Protein Assay Standards: Bovine Serum Albumin (BSA) Set, 7 × 3.5mL23212 Bovine Gamma Globulin Standard, 2mg/mL, 10 × 1mL ampules23213 Pre-Diluted Protein Assay Standards, (BGG) Set, 7 × 3.5mL aliquots23235 Pierce Micro BCA Protein Assay Kit, working range of 0.5-20µg/mL23236 Coomassie Plus (Bradford) Assay Kit, working range of 1-1500µg/mL23215 Compat-Able™ Protein Assay Preparation Reagent Set23250Pierce BCA Protein Assay Kit−Reducing Agent CompatibleAdditional InformationA.Please visit our website for additional information including the following items:•Frequently Asked Questions•Tech Tip protocol: Eliminate interfering substances from samples for BCA Protein AssayB.Alternative Total Protein Assay ReagentsIf interference by a reducing substance or metal-chelating substance contained in the sample cannot be overcome, try the Thermo Scientific Coomassie Plus (Bradford) Assay Kit (Product No. 23236), which is less sensitive to such substances.C.Cleaning and Re-using GlasswareExercise care when re-using glassware. All glassware must be cleaned and given a thorough final rinse with ultrapure water.D.Response characteristics for different proteinsEach of the commonly used total protein assay methods exhibits some degree of varying response toward different proteins. These differences relate to amino acid sequence, pI, structure and the presence of certain side chains or prosthetic groups that can dramatically alter the protein’s color response. Most protein assay methods use BSA or immunoglobulin (IgG) as the standard against which the concentration of protein in the sample is determined (Figure 1). However, if great accuracy is required, prepare the standard curve from a pure sample of the target protein.Typical protein-to-protein variation in color response is listed in Table 3. All proteins were tested at 1000µg/mL using the 30-minute/37°C Test Tube Protocol. The average net color response for BSA was normalized to 1.00 and the average net color response of the other proteins is expressed as a ratio to the response of BSA.Figure 1: Typical color response curves for BSA and BGG using the Standard Test Tube Protocol (37°C/30-minute incubation). Table 3. Protein-to-protein variation. Absorbance ratios (562nm) for proteins relative to BSA using Protein Tested Ratio Albumin, bovine serum 1.00 Aldolase, rabbit muscle 0.85 α-Chymotrypsinogen, bovine 1.14 Cytochrome C, horse heart 0.83 Gamma globulin, bovine1.11 IgG, bovine 1.21 IgG, human 1.09 IgG, mouse 1.18 IgG, rabbit 1.12 IgG, sheep1.17 Insulin, bovine pancreas 1.08 Myoglobin, horse heart0.74 Ovalbumin 0.93 Transferrin, human 0.891.02 Standard Deviation 0.15Coefficient of Variation14.7%Cited References1. Smith, P.K., et al. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem . 150:76-85.2. Wiechelman, K., et al. (1988). Investigation of the bicinchoninic acid protein assay: Identification of the groups responsible for color formation. Anal Biochem . 175:231-7.3. Kessler, R. and Fanestil, D. (1986). Interference by lipids in the determination of protein using bicinchoninic acid. Anal. Biochem . 159:138-42.4.Brown, R., et al. (1989). Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal. Biochem . 180:136-9.Product ReferencesAdilakshami, T. and Laine, R.O. (2002). Ribosomal protein S25 mRNA partners with MTF-1 and La to provide a p53-mediated mechanism for survival ordeath. J. Biol. Chem. 277:4147-51.Fischer, T., et al. (1999). Clathrin-coated vesicles bearing GAIP possess GTPase-activating protein activity in vitro. Proc. Nat. Acad. Sci. 96:6722-7. Prozialeck, W.C., et al. (2002). Chlamydia trachomatis disrupts N-cadherin-dependent cell-cell junctions and sequester β-catenin in human cervicalepithelial cells. Infection and Immunity 70:2605-13.Roberts, K.P., et al. (2002). A comparative analysis of expression and processing of the rat epididymal fluid and sperm-bound forms of proteins D and E.Biology of Reproduction 67:525-33.Triton ® is a registered trademark of Rohm & Haas Co.Brij ®, Tween ® and Span ® are registered trademarks of ICI Americas. Zwittergent ® is a registered trademark of American Hoechst Corporation.This product (“Product”) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as set forth in the Product documentation, specifications and/or accompanying package inserts (“Documentation”) and to be free from defects in material and workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to anyone other than the original purchaser of the Product (“Buyer”).No other warranties, express or implied, are granted, including without limitation, implied warranties of merchantability, fitness for any particular purpose, or non infringement. Buyer’s exclusive remedy for non-conforming Products during the warranty period is limited to replacement of or refund for the non-conforming Product(s).There is no obligation to replace Products as the result of (i) accident, disaster or event of force majeure, (ii) misuse, fault or negligence of or by Buyer, (iii) use of the Products in a manner for which they were not designed, or (iv) improper storage and handling of the Products.Current product instructions are available at /pierce . For a faxed copy, call 800-874-3723 or contact your local distributor. © 2011 Thermo Fisher Scientific Inc. All rights reserved. Unless otherwise indicated, all trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries. Printed in the USA.Table 2. Compatible substance concentrations in the BCA Protein Assay (see text for details).§* Diluted with ultrapure water.** Detergents were tested using high-purity Thremo Scientific Surfact-Amps Products, which have low peroxide content.-- Dashed-line entry indicates that the material is incompatible with the assay.§ For a more extensive list of substances, download Tech Tip # 68: Protein Assay Compatibility Table from our website. This Tech Tip includes compatible substances for all of our protein assays and enables easy comparisons.。

博迈德BCA试剂盒说明书产品简介蛋白质定量是蛋白质研究的基础工作之一。

博奥森开发的BCA蛋白质检测试剂(bicinchonicacid）是理想的蛋白质定量方法;是当前比Lowry法更优越的专用于检测总蛋白质含量的产品。

该方法以快速灵敏、稳定可靠，对不同种类蛋白质检测的变异系数非常小而倍受专业人士的青睐。

BCA法测定蛋白浓度不受绝大部分样品中的化学物质的影响。

在组织细胞裂解实验中，常用浓度的去垢剂SDS，TritonX-100，Tween 不影响检测结果，但受螯合剂(EDTA，EGTA、)还原剂(DTT巯基乙醇)和脂类的影响。

实验中，若发现样品稀释液或裂解液本身背景值较高，可试用Bradford蛋白浓度测定试剂盒。

该产品不受此影响，因此与之配合使用，可免除您实验中的许多烦恼。

基本原理碱性条件下，蛋白将Cu2+还原为Cu+，Cu+与BCA试剂形成紫颜色的络合物，测定其在562nm处的吸收值，并与标准曲线对比，即可计算待测蛋白的浓度。

主要特点1.准确XX线性XX:BCA试剂的蛋白质测定范围是10-2000ug/ml;2.快速:45分钟内完成测定，比经典的Lowry法快4倍而且更加方便。

3.经济实用:在微孔板中进行测定，可大大节约样品和试剂用量。

4.不受样品中离子型和非离子型去污剂影响。

5.检测不同蛋白质分子的变异系数远小于考马氏亮蓝。

包装及保存BCA试剂A70ml室温保存BCA试剂B1.4ml室温保存蛋白标准C0.5ml-20C保存(5mg/1mlBSA)本试剂盒自订购xx一年内有效。

注意事项1·在低温条件或长期保存出现沉淀时，可搅拌或37C温育使溶解，如发现细菌污染则应丢弃。

2.样品中若含有EDTA、EGTA、DTT硫酸铵、脂类会影响检测结果，请试用Bradford蛋白浓度测定试剂盒;高浓度的去垢剂也影响实验结果，可用TCA沉淀去除干扰物质。

3.更为精确的蛋白浓度结果，每个蛋白梯度和样品均需做复孔均应做标准曲线。

BCA蛋白定量试剂盒使用说明书一、产品简介BCA 蛋白定量试剂盒是一种常用的蛋白质浓度测定方法，具有操作简便、灵敏度高、准确性好等优点。

本试剂盒适用于细胞裂解液、组织匀浆、血清、血浆等多种样品中总蛋白浓度的定量测定。

二、试剂盒组成1、 BCA 工作液：A 液与 B 液按 50:1 的比例混合而成，现配现用。

2、蛋白标准品（2mg/ml）：牛血清白蛋白（BSA）溶液。

3、 96 孔板三、所需设备1、酶标仪（波长 562nm）2、移液器（量程分别为20μl、200μl、1000μl）3、涡旋振荡器4、离心机四、操作步骤1、标准曲线的绘制（1）将蛋白标准品（2mg/ml）用 PBS 或生理盐水稀释成一系列浓度梯度，如0μg/ml、25μg/ml、50μg/ml、100μg/ml、200μg/ml、400μg/ml、600μg/ml、800μg/ml、1000μg/ml、1500μg/ml、2000μg/ml。

（2）在 96 孔板中，每个浓度梯度设置 2 个复孔。

分别向孔中加入25μl 不同浓度的标准品溶液。

（3）向每个孔中加入200μl BCA 工作液，轻轻振荡混匀，37℃孵育 30 分钟。

（4）使用酶标仪在 562nm 波长下测定吸光度值（OD 值）。

（5）以蛋白浓度为横坐标，OD 值为纵坐标，绘制标准曲线。

2、样品测定（1）将待测样品用 PBS 或生理盐水适当稀释（若样品浓度过高，可能会超出标准曲线范围）。

（2）在 96 孔板中，设置样品孔和空白孔（仅加25μl 稀释液和200μl BCA 工作液）。

向样品孔中加入25μl 稀释后的样品。

（3）向每个孔中加入200μl BCA 工作液，轻轻振荡混匀，37℃孵育 30 分钟。

（4）使用酶标仪在 562nm 波长下测定样品孔和空白孔的吸光度值（OD 值）。

（5）样品的蛋白浓度可根据标准曲线计算得出。

五、注意事项1、 BCA 工作液应现配现用，避免长时间放置导致失效。

BCA蛋白浓度测定试剂盒说明23225蛋白质化验试剂盒：为500试管或5000微孔板的检测提供充足的试剂23227蛋白质化验试剂盒：为250试管或2500微孔板的检测提供充足的试剂试剂盒组分：BCA 试剂A，1000 mL (No. 23225产品中) 或500mL ( No. 23227产品中)，碳酸钠，碳酸氢钠，二喹啉甲酸，酒石酸钠溶于0.1 M氢氧化钠中。

BCA 试剂B , 25 mL, 包括4%硫酸铜一次性标准白蛋白, 2mg/ mL, 10 × 1 mL 安瓿, 包含2 mg/ mL牛血清白蛋白(BSA) 存在于0.9% 盐和0.05%叠氮化钠中。

储存：以上试剂保持在室温下储存和装运注意：如果试剂A 或试剂B 在低温下运输或长期储存时出现沉淀现象，可以通过缓慢加温或轻轻搅拌溶液使沉淀物溶解。

当试剂变色或确定微生物污染时请丢球试剂盒。

目录介绍 (1)准备标准试剂和工作试剂 (2)准备试管 (3)准备微型版 (3)故障检修 (4)有关美国热电其他产品 (5)附加信息 (5)参考文献 (6)介绍美国热电（Thermo）公司的BCA蛋白浓度测定试剂盒是基于二喹啉甲酸(BCA)通过比色检测和定量测定总蛋白的洗涤剂兼容配方。

该方法通过碱性介质中的一种蛋白结合了Cu2使其显著减少转变为Cu1 （缩二脲反应）。

用一种含二奎琳甲酸的试剂选择性的比色法高敏感的比色杯中的Cu1. 这种测定方法的紫色色反应产物是通过BCA的两个分子和亚铜离子螯合作用形成的。

这种水溶性复合物在562nm 处有强吸收峰。

在大的活性范围内(20-2000µg / mL）几乎同蛋白浓度增加呈线性关系。

BCA 法不是真正的终点的方法；也就是说，最终颜色继续发展。

孵化之后, 继续的颜色发展速度是足够慢以允许一起进行测定大量样本。

大分子结构的蛋白质,肽键的数量和存在的四个特定氨基酸(半胱氨酸,胱氨酸，色氨酸和酪氨酸)据说是与BCA形成颜色产物的原因。