Protocol for BelloCell-500E Operation

Sysmex CS-5100 - Application guidePage 1 of 19 Application guide for the Platelet function test by automated aggregation withEpinephrine (AG002K)This application guide describes the parameters and the procedure for measuring epinephrine-induced platelet aggregation, using the Epinephrine kit (AG002K) for optimal performance on Sysmex CS-5100. This application guide is based on the Sysmex CS-5100 system with the software version 00-13. This document is the only valid source of information for the assay application in the Sysmex CS-5100 system. HYPHEN BioMed has validated the instructions provided for the combination of reagents, instrument, software and customizable features of this system in order to optimize the performance of the product and comply with specifications. The modification of these parameters may affect performance and results. If the application is modified, the user shall be responsible for the validation of the modifications and their impact on all the results.For further information, please refer to the analyzer and reagents instruction manual. A specific configuration is required to perform the test.1. Test Principle:When added to platelet-rich plasma (PRP), epinephrine binds to the α2-adrinergic receptors on the platelet surface, inducing an initial wave of reversible aggregation. This primary aggregation leads to exposure of fibrinogen receptors and inhibition of adenylate cyclase activity. A second wave of aggregation, clearly distinct from the first, is triggered by ADP release from δ granules and by thromboxane A2 synthesis. Epinephrine is considered to be a weak agonist as the effects it produces are partial and reversible.2. Test synopsis:3. Reagents and material required but not provided:∙Distilled water∙Saline solution (0.9% NaCl)∙4mL Cup (424-1160-8)∙SB Cuvette (064-1041-9) and SB Kit tool (063-4151-5)∙Laboratory materials and equipment4. Preparation and stability of reagents:HYPHEN BioMed reagents). [3] It is recommended to transfer to 4 mL cup. [4] For multiple concentrations. Do not use a non-validated concentration.5. Recommendations:Homogenize well the reagents prior to each use. To obtain a homogeneous reactivity, it is essential to allow the temperature of the reagents stabilize for at least 30 minutes on board the analyzer prior to any use. Do not inter-change reagent vials from different batches.Quality control tests must be performed regularly, and with each change of reagent batch, after any major maintenance work on the analyzer, and when the results do not comply with the expected values for the method.The performance may vary slightly depending on the analyzer used. Each laboratory may establish its own acceptance range.6. Results:∙ The results are expressed in % of maximum aggregation. ∙It is recommended to make duplicate measurements.7. Performance and characteristics:less than 20% and the abnormal samples less than 30% for the maximum aggregation measurement (%).Aggregation curve with Epinephrine (For information only)Expected valuesThe values obtained on healthy subjects vary from one laboratory to another, each laboratory must determine its own reference ranges.The reference range was established on health adult subjects (n=150) using Sysmex CS-5100, 2000i, 2100i, 2400, 2500 (Central 90%, 97.5th percentile).The reference range measured is 68.8 – 99.8 % of maximum aggregation (median: 87.2% of maximum aggregation). SpecificityThe mean value measured out of 150 normal platelet-rich plasmas is 87.0 % of the maximum aggregation.exhibit different behavior because Intralipids and Interference Check.A Plus are artificial materials.Interference Studies have been established using the CS-2000i and 5100 System.[2]Platelet Poor Plasma (PPP) samples with high intralipids levels may give an error code [7030.0000.0000 / Ana_PPP_High1]. Accordingly, the associated result may give an error code [7030.0000.0000 / Ana_PPP_High1] and [7050.0000.0000 / Ana_Tube_Position].The cause of the error code is excess turbidity.[3]Refer to ISTH recommendation (Cattaneo, et al, 2013), "hemolyzed samples may affect platelet aggregation and should be discarded."Comparison of Methods:The comparison of the methods was established using a CS-2000i system and confirmed for the CS-5100 system.8. Setup of test:The special position may contain a maximum of 48 (24x2) cuvettes with magnetic stir bar (SBC) to be inserted with the SBC tool.Place the PPP and PRP on the sample rack side by side in the order PPP and then PRP. Thus, the PPPs will be in odd numbered positions while the PRP will be in the even numbered positions. Touch the agonist button to start the measurement on the PRP- Once the PPP and PRP analysis has ended, the instruments calculates the percentage of maximum aggregation (% Max aggregation) automatically.Assay Group Setup-[PPP]▲ [Main Menu] - [Settings] - [Assay Group Settings] - [PPP]► [Basic][1][1] It is recommended to perform the tests in duplicate.►[Re-analysis]NOTE: Please adjust the parameters of the Re-analysis conditions, if necessary.► [Reflex]NOTE: This tab allows defining the Reflex options. Please adjust the parameters of the Reflex conditions, if necessary.►[QC]NOTE: This tab allows defining the QC options. Please adjust the parameters of the QC conditions, if necessary. ►[Test Protocol]Assay Parameter Setup- [PPP]▲ [Main Menu] - [Settings] - [Assay Group Settings] - [PPP] - [Basic] - [PPP]►► [Data Check]►[Evaluation Preset]▼▼ [Evaluation Check Parameter]▼Assay Group Setup-[Epi]▲ [Main Menu] - [Settings] - [Assay Group Settings] - [Epi]► [Basic][1][1] It is recommended to perform the tests in duplicate.►NOTE: Please adjust the parameters of the Re-analysis conditions, if necessary.► [Reflex]NOTE: This tab allows defining the Reflex options. Please adjust the parameters of the Reflex conditions, if necessary. ►[QC]NOTE: This tab allows defining the QC options. Please adjust the parameters of the QC conditions, if necessary.►Assay Parameter Setup- [Epi_min]▲ [Main Menu] - [Settings] - [Assay Group Settings] - [Epi] - [Basic] - [Epi_min]► [Calculation Method]►►[Evaluation Preset]▼ [Evaluation Parameter]▼▼ [Research Parameter]Assay Parameter Setup- [Epi_s]▲ [Main Menu] - [Settings] - [Assay Group Settings] - [Epi] - [Basic] - [Epi_s]►► [Data Check]►[Evaluation Preset]▼▼ [Evaluation Check Parameter]▼Assay Parameter Setup- [Epi_e]▲ [Main Menu] - [Settings] - [Assay Group Settings] - [Epi] - [Basic] - [Epi_e]► [Calculation Method]►►[Evaluation Preset]▼ [Evaluation Parameter]▼▼ [Research Parameter]Formula setting - [Epi%]FormulaManagement ID Assay Parameter Formula18720 Epi% (([Epi_s]-[ Epi_min])/([Epi_s]-[PPP]))*10018721 Epi2% (([Epi2_s]-[ Epi2_min])/([Epi2_s]-[PPP]))*10018722 Epi3% (([Epi3_s]-[ Epi3_min])/([Epi3_s]-[PPP]))*10018723 Epi4% (([Epi4_s]-[ Epi4_min])/([Epi4_s]-[PPP]))*10018724 Epi5% (([Epi5_s]-[ Epi5_min])/([Epi5_s]-[PPP]))*100。

AS1490操作说明（本说明仅供参考，请以英文原版操作手册为准）用组织研磨机（机械珠磨破碎）进行预处理用户需提供的材料• 珠打装置(如MP Biomedicals FastPrep®-24仪器)• 无菌，防回吸移液枪头• 微型离心机或平板专用离心机1. 在每个离心管或孔的底部放置至多20mg的叶片组织。

2. 把一个珠子(或些许珠子，根据制造商的建议)放入每个管或孔。

3. 每个离心管或孔中加入300ul Tail Lysis Buffer (TLA)。

4. 每孔加入10ul RNase A(可选)。

注:如处理大量样品，使用前应立即准备足量的Tail Lysis Buffer和RNase A，将 310ul上述混合物加到每个样品中。

5. 使用制造商推荐的时间和速度运行珠打装置。

可能需要进行一些优化。

6. 将需要提取的离心离心管或板子放入离心机中，以最大速度旋转2分钟，以去除任何固体颗粒。

7. 制备Maxwell® RSC 样本制备托盘和试剂条(下一页制备样本托盘)。

8. 在每个Maxwell® RSC Plant DNA试剂条的第1孔中加入300ul无核酸酶水。

(1号孔是离试剂盒编码端最近、离用户最远的孔)。

9. 将每个植物裂解物样本从提取管或板子孔中转移到Maxwell® RSC试剂条的第1孔中。

只转移透明液体，注意不要转移任何固体物质到试剂条内。

用EP管、杵和液氮进行预处理用户需提供的材料• 颗粒杵(Sigma Aldrich Cat. # Z359947)• ClickFit EP管，1.5ml• 液氮• 无菌，防回吸移液枪头• 微型离心机1. 在1.5ml ClickFit EP管底部放置至多20mg的叶片组织。

2. 向植物组织样品中加入液氮。

让液体蒸发，冷冻样品。

3. 使用颗粒杵，尽可能彻底的对管壁研磨冷冻植物组织。

4. 每管加300 ul Tail Lysis Buffer (TLA)5. 每管加入10 ul RNase A(可选)。

The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.Lab Water SolutionsWater Purification Solutions for Clinical AnalyzersSimplify seamless performance.Secure the performance of clinical analyzers with water purification excellenceWater contamination can interfere with clinical assays, potentially causing errors, unstable results, delays and downtime. Patented and robust Milli-Q ® water purification technologies ensure that clinical analyzersare fed with water that is contaminant free.• Remove ions and organic contaminants to trace levels Q-Gard ®/IPAK Quanta ® polishing cartridges • Prevent contamination of circulating water• UV lamp• Opticap ®/Millipak ® 0.22 μm sterilizing filtersBacteria and their by-products are a major source of water contamination that can impact clinical assays results. Bacterialcontamination can come from tap water and air , resulting in bacterialgrowth in analyzer reservoirs, tubing and water baths.To manage a continuously growing number of tests in a timely manner, clinical labs need to be backed by innovative and reliable technologies.2• Highly reliable systems.Fulfill both daily volume requirements and cover peak-use periods with robust Milli-Q ® systems, manufactured in a Merck site that has been ISO 9001:2015 and ISO 14001 certified.• Emergency solutions.In case a system is non-operational, the emergency bypass procedure is easy to set up. Available on Milli-Q ® CLX 7000 systems.With Elix ® EDI technology, deionization quality and costremain constant, and are independent of feed water quality, RO membrane efficiency, and pure water use.Total traceability of water quality.Milli-Q ® water purification systems automatically record all water quality parameters, volumes and any system events directly in the system. Events include alerts, setting modifications, purification cartridgereplacements, service activities, and more. This is especially critical for clinical laboratories, as water quality must be documented for labs seeking to conform to CLSI ® and accreditation (or reaccreditation) to the ISO 15189:2012 standard supported by CAP 15189SM accreditation.• Water quality parameters (resistivity,temperature, and TOC) are rapidly searchable and graphable over any specified timeline.†• An Event Traceability tool † makes it easy to view events by type and over a specified timeline. Planning for future maintenance is supported as past and future actions are clearly displayed on this interactive MyMilli-Q™ tool.• E-record archiving facilitates accreditation as all data generated are automatically stored in the system memory.Past and future maintenance actions are clearly displayed on the interactive MyMilli-Q™ Event Traceability tool.†Available with subscription to MyMilli-Q™ remote services.CAP , College of American Pathologists; CLSI, Clinical and Laboratory Standards Institute; TOC, total organic carbon.3• Simplified, tutorial-assisted cartridge changes . There’s no need to open the water system tochange purification cartridges. System care is rapid and can usually be managed in house following on-screen guides. RFID technology ensures automatic traceability of cartridges and prevents the insertion of an incorrect consumable.• Walk-away filling . There’s no need to stand in front of the water system to feed a 5- or 10-L analyzer reservoir. Volumetric Dispense mode on our patented E-POD ® and Q-POD ® dispensers fill containers to the set volume.Fast and simple water systemmanagement—any time, anywhereMaintenance is simplified by digital design.Decreasing the time spent maintaining or monitoring lab water systems can save precious time and stress, and allow greater focus on more important operations and urgencies. Milli-Q ® water purifications systems’ innovative technologies,designs and digital services contribute to a more efficient working day.Quick and easy cartridge changes with our patented pack-locking system and on-screen, step-by-step instructions.4Efficient assistance worldwide with unique digital service capabilities.• Fast and remote water system access.MyMilli-Q™ remote services allows for:-Secure, real-time monitoring of water systemstatus and water quality data-Remote control of water systems-Automatic notifications by email or SMS in case ofalerts and alarmsMyMilli-Q™ remote services gives youconfidence that clinical analyzers arebeing fed with the correct water quality.• Accelerate analyzer troubleshooting. TheMyMilli-Q™ troubleshooting portal helps yourAnalyzer Hotline and clinical labs to quicklydetermine a clear water system status for the• Quickly find consolidated, online contractinformation for all systems in all facilities.The MyMilli-Q™ customer portal simplifies facilitiesmanagement and remote system care as you’ll beable to:-Manage consumable deliveries-Schedule maintenance visits-Renew service plans-Streamline audit preparation with rapid access toeasily traceable service histories and reports• Experience care-free water system operation.Milli-Q® systems require minimal, once-a-yearmaintenance and are backed by a reliable,reactive and knowledgeable service team in everygeography. Preventive maintenance is performedby certified field service engineers who provide usertraining and following auditable Standard OperatingProcedures.* Available for Milli-Q® CLX 7000 systems that feed analyzers.5Integrate scalability and flexibility into laboratory workflowsAttain optimal laboratory designs with expert advice.Consult with our water experts for building project management advice that is adapted to local water quality, lab setting and space constraints. Your local Merck team has a depth of engineering and project managementcapabilities, and can design and customize lab installations to exactly meet the analyzer’s requirements.Analyzer ApplicationVolumeAnalyzer feedWater qualityMolecular biology,LC-MS, ICP-MS, HPLC Histology, Cytology,Microbiology Water requiredManualUltrapure / Type 1Pure / Type 2 / CLRWAutomatedHematology,HemostatisImmunochemistry,Biochemistry,Modular analysesDefine and design your total waterpurification solution in collaboration with our Commercial Engineering and Design team.Reduce environmental impact of clinical labs• Up to 50% water savings.Reject RO water is recycled by E.R.A.® technology, reducing water use up to 50% vs. other reducing waste.• Reduced chemical waste.Elix ® EDI technology eliminates the need for hazardous chemicalTop 1% for Sustainability Management.companies assessed.6Bringing the right fit with a portfolio of compact systems.Our comprehensive range of water purification systems and customizable solutions can be adapted to fit laboratory needs. No matter the size, set up, or quality requirements, solutions are available that produce from a few liters to up to several thousand liters per day of pure, Clinical Laboratory Reagent Water (CLRW), and ultrapure quality water.Water quality Feed toanalyzer Max. dailyvolume System range System water specificationsCLRW Automated3000 L Milli-Q® CLX 7000Resistivity > 15 MΩ·cm @ 25°CBacteria < 1 cfu/mLTOC < 30 ppbFiltration 0.22 μm Automated300 L AFS®, AFS® D/EPure (Type 2) water Automated9000 L Milli-Q® HX 7000 SDMilli-Q® HX 7000Resistivity > 5 MΩ·cm @ 25°C,typically 10-15Bacteria < 10 cfu/mL(1)TOC < 30 ppbManual2 300 L Milli-Q® IX7003/05/10/15Ultrapure (Type 1) water Manual2300 L Milli-Q® IQ7000/03/05/10/15Resistivity 18.2 MΩ·cm @ 25°C Bacteria ≤ 0.01 cfu/mL (≤ 10 cfu/L)(3) TOC typically ≤ 5 ppbNo particles with size > 0.22 μm(4)CLRW, clinical laboratory reagent water; TOC, total organic carbon.1. For Milli-Q® IX, bacteria ≤ 0.01 cfu/mL (≤ 10 cfu/L) with Millipak®, Millipak® Gold or Biopak® filter when installed and used in a laminar flow hood.2. With semi-automated volumetric dispensing.3. With Millipak®, Millipak® Gold or Biopak® filter when installed and used in a laminar flow hood.4. With Millipak® or Millipak® Gold filter.Milli-Q® IX systems deliver consistent-quality pure(Type 2) water with both manual with semi-automatedvolumetric dispensing.Milli-Q® products are for laboratory use only and are not medical li-Q® CLX 7000 systems can deliver up to 3000 L/day of CLRW to analyzers by automated feed.7Lab Water Solutions© 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved. Merck, the vibrant M, Milli-Q, MyMilli-Q, Elix, E.R.A., Millipak, Biopak, IPAK Gard, IPAK Quanta, Q-Gard, Opticap, Progard, E-POD, Q-POD and AFS are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.Lit. No. MK_BR8085ENFor more information, please visit our website:/LabWater。

武汉普诺赛生命科技有限公司 Procell Life Science&Technology Co.,Ltd.使用前请仔细阅读说明书。

如果有任何问题，请通过以下方式联系我们：全国免费电话：400-650-3656销售电话：************销售邮箱：*****************.cn官方网站：CL-0493BV2（小鼠小胶质细胞）武汉普诺赛生命科技有限公司——您身边的细胞培养专家Copyright ©2020-2021 Procell Life Science&Technology Co.,Ltd. All Rights Reserved 细胞株培养扩增技术服务申明本公司受贵单位委托，进行细胞株的技术服务工作，并收取相应细胞株技术服务费用，细胞株技术服务具体项目清单见订购合同。

本公司提供完善的技术支持及售后服务，收到产品后处理方式及相应售后条款参见《细胞售后条例》。

收到常温细胞后如何处理？（细胞培养详细操作步骤请参照《普诺赛细胞培养操作指南》）1. 收到常温细胞后，及时拍照记录有无漏液/瓶身破损现象。

2. 用75%酒精擦拭细胞培养瓶表面，显微镜下观察细胞状态。

先不要打开培养瓶盖，将细胞置于细胞培养箱内静置培养2-4小时，以便稳定细胞状态。

3. 仔细阅读细胞说明书，了解细胞相关信息，如贴壁特性（贴壁/悬浮）、细胞形态、所用基础培养基、血清比例、所需细胞因子、传代比例、换液频率等。

4. 静置完成后，取出细胞培养瓶，镜检、拍照，记录细胞状态（所拍照片将作为后续服务依据）；建议细胞传代培养后，定期拍照、记录细胞生长状态。

5. 管内离心，吸去上清，管底沉淀加入80%，可接回原瓶培养，若大于80%6. 培养瓶或用移液器轻轻吹打使其脱落，若通过吹打的方式不易脱落的细胞则需要用胰蛋白酶消化后收集细胞传代。

7. 注意事项有疑问的，可跟我们的技术支持交流。

MSD GOLD™ SULFO-TAG NHS-EsterMSD GOLD SULFO-TAG™ NHS-Ester 150 nmol (sufficient for conjugating 1 mg of IgG) R91AO-12 µmol (sufficient for conjugating 10 mg of IgG) R91AO-2MSD GOLD SULFO-TAG NHS-Ester Conjugation Packs Pack 1 (sufficient for conjugating 5 x 200 µg of IgG) R31AA-1 Pack 2 (sufficient for conjugating 5 x 1 mg of IgG) R31AA-2MSD Labeling ReagentsMSD GOLD SULFO-TAG NHS-EsterFor labeling aminesNote:150 nmol size of MSD GOLD SULFO-TAG NHS-Ester is sufficient for conjugating 1 mg of IgG and the 2 µmol size is sufficient for conjugating 10 mg of IgG at a challenge ratio of 20.Each MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack contains enough material for 5 reactions. At a challenge ratio of 20, Pack 1 is sufficient for conjugating 200 µg of IgG per reaction, and Pack 2 is sufficient for conjugating 1 mg of IgG per reaction.This package insert must be read in its entirety before using this product.FOR RESEARCH USE ONLY.NOT FOR USE IN DIAGNOSTIC PROCEDURES.MESO SCALE DISCOVERY®A division of Meso Scale Diagnostics, LLC.1601 Research BoulevardRockville, MD 20850-3173 USAMESO SCALE DISCOVERY, MESO SCALE DIAGNOSTICS, MSD, MSD GOLD, DISCOVERY WORKBENCH, MULTI-ARRAY, MULTI-SPOT, QUICKPLEX, SECTOR, SECTOR PR, SECTOR HTS, SULFO-TAG, U-PLEX, V-PLEX, S-PLEX, STREPTAVIDIN GOLD, MESO, , SMALL SPOT (design), 96 WELL 1, 4, 7, & 10-SPOT (designs), 384 WELL 1 & 4-SPOT (designs), MSD (design), U-PLEX (design), V-PLEX (design), S-PLEX (design), and SPOT THE DIFFERENCE are trademarks and/or service marks of Meso Scale Diagnostics, LLC. ©2015 Me s o Scale Diagnostics, LLC. All rights reservedTable of ContentsIntroduction (4)Preparation of MSD GOLD SULFO-TAG Conjugates (5)MSD GOLD SULFO-TAG NHS-Ester Conjugation Packs (6)Additional Materials and Equipment (6)Protocol (8)Storage, Handling, and Stability (10)FAQs (11)Worksheet (14)Ordering InformationMSD Customer ServicePhone: 1-240-314-2795Fax: 1-301-990-2776Email: CustomerService@MSD Scientific SupportPhone: 1-240-314-2798Fax: 1-240-632-2219 attn: Scientific SupportEmail: ScientificSupport@IntroductionThis protocol details the conjugation procedure for proteins of molecular weight (MW) > 40,000 D altons using MSD GOLD SULFO-TAG NHS-Ester label. The straightforward procedure involves an optional buffer exchange step, a 2-hour incubation step, and a mandatory buffer exchange step to quickly isolate the conjugated protein using a spin column. MSD GOLD SULFO-TAG NHS-Ester (Figure 1) is an amine reactive, N-hydroxysuccinimide ester which readily couples to primary amine groups of proteins under mildly basic conditions to form a stable amide bond.MSD GOLD SULFO-TAG conjugates are stable and may be used at low concentrations. These features minimize time, cost, and labor as large batches of a stable conjugate can be prepared, validated, and used for long periods of time. Its excellent performance characteristics and simple conjugation procedure make MSD GOLD SULFO-TAG NHS-Ester the product of choice for molecules that contain primary amines (e.g., lysine-containing proteins). MSD GOLD SULFO-TAG offers low non-specific binding, resulting in highly sensitive detection when used in conjunction with MSD instruments.Figure 1: MSD GOLD SULFO-TAG NHS-EsterPreparation of MSD GOLD SULFO-TAG ConjugatesGeneral NotesIn order to minimize hydrolysis of MSD GOLD SULFO-TAG NHS-Ester, the reagent should be dissolved in cold distilled water just prior to its addition to the protein solution. If necessary, the stock MSD GOLD SULFO-TAG NHS-Ester solution can be kept on ice for up to 10 minutes. The reconstituted solution is unstable and any unused material should be discarded. Consider conjugating more than one protein at the same time to maximize the use of the MSD GOLD SULFO-TAG NHS-Ester reagent. Generally, 150 nmol of MSD GOLD SULFO-TAG NHS-Ester is sufficient for conjugation of up to 1 mg of IgG.The labeling ratio is the number of molecules of MSD GOLD SULFO-TAG conjugated to each molecule of protein. Optimal conjugation ratios for a MSD GOLD SULFO-TAG conjugated protein should be determined empirically for each specific application. For most applications using IgG antibodies (MW ~150,000), optimal performance is obtained with conjugation ratios between 2:1 and 20:1. In this range, assay signals typically show a linear dependence on conjugation ratio. Conjugation ratios significantly higher than 10:1 can be counterproductive and may lead to elevated background signals or loss of binding activity. For proteins that are significantly smaller than IgGs, lower conjugation ratios (between 1:1 and 5:1) may provide better assay performance.The challenge ratio is the number of moles of MSD GOLD SULFO-TAG per mole of protein in the conjugation reaction mixture. The challenge ratio required to achieve a specific conjugation ratio depends on a number of factors including pH, temperature, protein concentration, protein size, and the number of lysines available for coupling. Conjugating a 2 mg/mL IgG solution using the standard conditions described in this protocol will typically result in a label incorporation of approximately 50% (i.e., a challenge ratio of 10:1 will result in a conjugation ratio of about 5:1). Conjugation efficiencies for other proteins may be different. In general, conjugating with high protein concentrations of 1–2 mg/mL in slightly alkaline PBS (pH 7.9) in the absence of preservatives yields the best conjugation efficiencies. Maintaining consistent conjugation conditions (protein concentration, buffer type, MSD GOLD SULFO-TAG NHS-Ester concentration, incubation time, and temperature) is important when preparing multiple batches of conjugated protein in order to achieve consistent assay results.When developing immunoassays, MSD recommends conjugating antibodies using the standard conditions outlined in this document and challenge ratios of 6:1, 12:1, and 20:1 to identify optimal conjugation conditions. If evaluating different conditions is not possible due to limited reagent quantities, a challenge ratio of 20:1 will generally provide good performance. For immunogenicity applications where an antibody drug or protein therapeutic is used, the suggested challenge ratios are 12:1 and 6:1 MSD GOLD SULFO-TAG:drug. If only one ratio is tested, a 10:1 challenge ratio is recommended. For details on building immunogenicity assays, please refer to the Bridging Immunogenicity Guidelines for Assay Development at . The protocol describes the MSD GOLD SULFO-TAG conjugation procedure for proteins with a MW > 40,000 D a. Smaller proteins/polypeptides may also be conjugated using MSD GOLD SULFO-TAG NHS-Ester as long as they have an accessible lysine or the N-terminal amino group; however, alternative separation methods may be needed to remove unconjugated label. MSD offers a variety of services for the custom conjugation of reagents including proteins, peptides, and non-proteinaceous molecules.MSD GOLD SULFO-TAG NHS-Ester Conjugation PacksMSD offers all-inclusive conjugation packs that include the components and guidance that may be necessary for conjugating and purifying detection reagents with MSD GOLD SULFO-TAG label. Two sizes of conjugation packs are offered: MSD GOLD SULFO-TAG Conjugation Pack 1 and Pack 2. The two packs enable the conjugation of different amounts of IgG. MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack 1 contains materials for conjugation and purification of up to 200 µg of IgG per reaction, and MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack 2 allows conjugation of up to 1 mg of IgG per reaction. Each pack contains enough material for 5 reactions (i.e., 5 vials of MSD GOLD SULFO-TAG NHS-Ester).Table 1. Components of MSD GOLD SULFO-TAG Conjugation Packs*MSD GOLD SULFO-TAG Conjugation Pack 1 includes 10 columns of 0.5 mL capacity and 10 syringes of 1 mL size, and MSD GOLD SULFO-TAG Conjugation Pack 2 includes 10 columns of 5 mL capacity and 10 syringes of 3 mL size.**The pH of Conjugation Buffer is stable for up to 2 weeks after opening the bottle. For long term use, it is recommended to readjust the pH of the solution.Additional Materials and EquipmentThe following additional materials may be required. Some items (denoted with a *) are included in the MSD GOLD SULFO-TAG Conjugation Packs.1.Conjugation Buffer* (Phosphate-buffered saline (PBS), pH 7.9, preservative-free)2.Conjugate Storage Buffer* (PBS pH 7.4 + 0.05% Sodium Azide)3.Polypropylene microfuge tubes4.Spin columns.* MSD recommends the use of Zeba Spin Desalting Columns, 40K MWCO of various sizes from ThermoScientific, Catalog # 87766–877735.15 mL conical tubes for use with Zeba Spin Desalting Columns, 40K MWCO, 2 mL column size6.Protein assay such as BCA, Bradford, or Lowry7.MSD Blocker A (optional), Catalog # R93AA-2 (250 mL) and R93AA-1 (1 L)8.Spectrophotometer capable of an OD455 measurementReagent Storage Size Quantity DescriptionMSD GOLD SULFO-TAG NHS-Ester ≤-70°C 150 nmol 5 vials MSD GOLD SULFO-TAG NHS-Ester label forcoupling to antibodies and other proteinsZeba Spin Desalting Column, 40K MWCO* 2–8°C 0.5 mL or5 mL10 columns Size exclusion chromatography columns for thepurification of proteins larger than 40,000 DaFilter, 0.22 µm RT N/A 10 each Filter for use during purification Syringe * RT N/A 10 each Syringe for use during purificationConjugation Buffer** 2–8°C 40 mL 1 bottle Phosphate-buffered saline (PBS), pH 7.9, preservative-freeConjugate Storage Buffer RT 40 mL 1 bottle PBS pH 7.4 + 0.05% Sodium Azide9. 0.2 µm filter* (optional) Table 2. Suggestions for 0.2 µm filter10. Concentrator (optional) Table 3. Suggestions for concentratorsNoteThe following table lists the catalog numbers of the Zeba Spin Desalting Columns, 40K MWCO, from Thermo Scientific and the recommended sample volume for each column.Table 4. Catalog numbers of Zeba Spin Desalting Columns from Thermo ScientificVendorCatalog # Volume of Conjugated ProteinWhatmanAV125EAQU ≥ 2.0 mL Millipore or Fisher Scientific (MILLEX-GV)SLGV004SL0.2–1.0 mLVendorCatalog # Range Millipore BIOMAX-50 concentrator, 50 MWCO UFV5BQK25 0.05–0.5 mL AMICON Ultra-4 concentrator, PLQK Ultracel-PL Membrane, 50 MWCOUFC805008 0.5–4.0 mL AMICON Ultra-15 concentrator, PLQK Ultracel-PL Membrane, 50 MWCOUFC9050240.5–15.0 mLThermo ScientificCatalog #Number of Columns/packZEBA Spin Desalting Column VolumeRecommended Volume of the Conjugation Reaction87766 25 0.5 mL 70–100 µL 87767 50 0.5 mL 70–100 µL 87768 5 2 mL 200–450 µL 87769 25 2 mL 200–450 µL 87770 5 5 mL 300–1,000 µL 87771 25 5 mL 300–1,000 µL 87772 5 10 mL 1,000–2,000 µL 877732510 mL1,000–2,000 µLProtocol1.Prepare a 1–2 mg/mL solution of the protein to be conjugated in Conjugation Buffer. Antibodies in a storage buffer withpreservatives such as sodium azide or EDTA must be buffer exchanged before the conjugation reaction. It is recommended that dilute protein solutions be concentrated to at least 1 mg/mL. Protein solutions should be concentrated and/or buffer exchanged using the spin columns described above or an alternative centrifugal filtration/concentration unit that has been equilibrated with preservative-free PBS, pH 7.9. (Use Conjugation Buffer when using MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack.) Filter the protein using a 0.2 µm filter. The concentration of the protein solution to be conjugated should be confirmed prior to beginning the conjugation reaction.Note: Conjugation buffer, 0.2 µm filter, and syringes are included in the MSD GOLD SULFO-TAG NHS-Ester Conjugation Packs.2.Equilibrate the protein to be conjugated with MSD GOLD SULFO-TAG NHS-Ester at the conjugation temperature of 23°C.A temperature range of 20°C to 25°C is acceptable. The equilibration can take between 10-30 minutes depending on thevolume of protein.3.Calculate the amount of MSD GOLD SULFO-TAG NHS-Ester stock solution required for the conjugation reaction using theformula depicted below and on the attached worksheet.EXAMPLE❑500 µL of 2 mg/mL antibody❑12:1 challenge ratio❑MSD GOLD SULFO-TAG stock = 3 nmol/µL1000 x 2 mg/mL x 12 x 500 µL = 80 nmol of MSD GOLD SULFO-TAG reagent required150,000 Da80 nmol of SULFO-TAG reagent = 26.7 µL of SULFO-TAG stock solution required for conjugation reaction3 nmol/µL SULFO-TAG stock solution4.Centrifuge the MSD GOLD SULFO-TAG NHS-Ester vial by pulse spinning for 1 minute or gently tap on a soft surface inorder to collect lyophilized material at the bottom of the vial. Reconstitute MSD GOLD SULFO-TAG NHS-Ester immediately prior to use with cold distilled water. For the 2 µmol and 150 nmol sizes of MSD GOLD SULFO-TAG NHS-Ester, dissolve with 200 µL and 50 µL, respectively to generate stock solutions of 10 and 3 nmol/µL. Gently vortex the vial to ensure complete dissolution of all lyophilized material.Note: Reconstituted MSD GOLD SULFO-TAG NHS-Ester may be kept for up to 10 minutes on ice prior to use.5.Add the calculated volume (from Step 3) of reconstituted MSD GOLD SULFO-TAG NHS-Ester to the protein solution andvortex immediately. Discard any remaining unused MSD GOLD SULFO-TAG NHS-Ester.6.Incubate at 23°C for 2 hours; a temperature range of 20°C to 25°C is acceptable. Shield the reaction from light bycovering the tube with aluminum foil or placing it in a dark area (e.g., a closed drawer). Take care to maintain consistent conjugation conditions between multiple preparation lots to ensure conjugation reproducibility.7.Prepare Zeba Spin D esalting Columns, 40K MWCO, towards the end of the incubation period. Remove the column'sbottom closure and loosen the cap. Do not remove the cap. Place the column in a collection tube to remove the storage buffer and wash the column 3 times with Conjugate Storage Buffer. Each preparation step should be carried out by centrifuging the columns, and their respective collection tubes, in a centrifuge with a swinging bucket rotor at 2–8°C.Refer to Table 5 below for the recommended sample volume, wash buffer volumes, collection tube sizes, and centrifugation times for each preparation step.Note: Reaction volumes larger than the capacity of a Zeba column should be distributed over multiple Zeba columns.8.Apply the conjugation reaction to the Zeba column in a dropwise manner following the recommendations in Table 2. Usinga swinging bucket rotor, centrifuge the columns in clean new collection tubes to purify the MSD GOLD SULFO-TAGconjugated protein. The MSD GOLD SULFO-TAG conjugated protein will be captured in the conical tubes. Retain the conjugated material in the conical tubes, and discard the columns.Note: The unconjugated MSD GOLD SULFO-TAG reagent will appear as yellow in the spin column.Table 5: Specifications for Zeba Spin Desalting Columns, 40K MWCOSize of Column (mL) 0.5 2 5 10 Sample Volume Range (µL) 70–100 200–450 300–1,000 1,000–2,000 Wash Buffer Volume 300 µL 1 mL 2.5 mL 5 mL Sample Volumes to use a stacker1N/A <350 µL <750 µL <1,500 µL Optional Stacker Volume N/A 40 µL 100 µL 200 µL Centrifugation Speed 1,500 x g1,000 x g1,000 x g1,000 x gCentrifugation Time(Min) Storage Solution Removal 1 2 2 2 Wash 1 1 4–8 4–8 4–8 Wash 2 1 2–8 2–8 2–8 Wash 32 3 5–8 5–8 5–8 Sample Recovery33–4 6–8 6–8 6–8addition of the protein to the column.2 If column is not mostly white after the third wash, the column may be spun for an additional 1–3 minutes.3 Additional sample recovery spin time is allowed if needed to ensure maximum recovery of sample. Overdrying the resin at this stage will not harm the protein; therefore, spinning for up to 8 minutes is allowed.9.It is recommended to filter the conjugated protein using a 0.2 µm filter. Filtration may cause some loss of the protein.Please refer to page 7 for the recommended filter units.10.Determine the molar protein concentration of the conjugated protein using a standard colorimetric protein assay such asBCA, Bradford, or Lowry.absorbance reading as MSD GOLD SULFO-TAG will absorb light at this wavelength.Note: Do not use an OD28011.Measure the absorbance of the MSD GOLD SULFO-TAG protein conjugate at 455 nm using a spectrophotometer. Dividethe measured value by the path length in cm, and then divide by the extinction coefficient of the label (15,400 M-1cm-1) to obtain the MSD GOLD SULFO-TAG label concentration in moles per liter. For reference, a formula calculation worksheet page is attached.12.To calculate the MSD GOLD SULFO-TAG label:protein conjugation ratio, divide the MSD GOLD SULFO-TAG labelconcentration value determined in step 11 by the molar protein concentration value determined in step 10.13.Antibody conjugates are usually stable for at least 2 years at 2–8°C at a concentration of 1–2 mg/mL; stability of otherprotein types should be determined. Conjugated proteins may be stored frozen at ≤-20°C or ≤-70°C, as long as the protein is stable to freeze-thaw cycles or stored in single-use aliquots. MSD GOLD SULFO-TAG conjugated proteins may be sensitive to extended exposure to light and should be stored in the dark or in amber or opaque vials. Short-term exposure of conjugates to light when carrying out assays is not a concern. If the protein concentration is low (< 0.1 mg/mL), consider adding a carrier protein such as 0.1% MSD Blocker A.Storage, Handling, and StabilityMSD GOLD SULFO-TAG NHS-Ester is supplied as a dry orange-red lyophilized solid. The vials should be stored at ≤-70°C. The expiration date of the product is indicated on the label. Following reconstitution, any remaining unused material should be discarded.FAQs1)What chemicals interfere with MSD GOLD SULFO-TAG conjugation?Primary amines and strong nucleophiles interfere with MSD GOLD SULFO-TAG NHS-Ester conjugation. Common reagents that can interfere with the amine coupling of NHS chemistry are:a)Trisb)Glycinec)Histidined)Azidee)Imidazolef)Glutathioneg)Ammonium ionsh)Glycerol2)What are typical carrier proteins in antibody solutions?a)BSAb)GelatinAntibodies should be obtained in carrier protein-free formulations for labeling with MSD GOLD SULFO-TAG NHS-Ester.Carrier proteins will interfere with MSD GOLD SULFO-TAG NHS-Ester conjugation and cannot be removed with desalting columns.3)What is the minimum amount of material that can be conjugated?Generally, 50-100 µg can be conjugated in PBS (without interfering buffer components) if the protein concentration is high enough (1–2 mg/mL). Otherwise, microconcentrators may be used to concentrate the antibody solution following equilibration of the microconcentrator with PBS.4)Are there alternatives to using Thermo Scientific Zeba Spin D esalting Columns for purifying the MSD GOLDSULFO-TAG conjugated antibody after conjugation?a)Users may purchase commercially available G-50 SEPHADEX columns or prepare G-50 SEPHADEX columns atthe bench. However, some G-50 columns may not be efficient in complete removal of unconjugated material. TheSEPHADEX grade is important. MSD recommends using fine grade SEPHADEX for preparing self-packed gelfiltration columns. Medium Grade SEPHADEX does not provide suitable separations and Superfine SEPHADEXdoes not allow an adequate flow rate without use of a pump. It is not recommended to use PD10 columns or G-25 SEPHADEX spin columns for purification of MSD GOLD SULFO-TAG-conjugated protein as these are notable to separate free MSD GOLD SULFO-TAG reagent from labeled conjugates.b)Alternatively, CENTRICON concentrators or similar microconcentrator products with adequate MWCO (forconcentrator information please refer to page 7) can be used to remove unbound label. Resuspend theconjugation mixture in a larger volume of PBS-0.05% azide, concentrate to a smaller volume, and then repeat theprocess as per the product instructions for desalting applications.c)Post-conjugation purification of proteins with MW < 40,000 Da will require alternative procedures (such as high-resolution size exclusion chromatography, HPLC, FPLC, etc.) because ZEBA Spin Desalting Columns or G-50columns will not provide adequate separation in this size range.5)What is the molecular weight of MSD GOLD SULFO-TAG?Unreacted MSD GOLD SULFO-TAG NHS-Ester has a molecular weight of 1,141 g/mol. After the conjugation reaction, each conjugated MSD GOLD SULFO-TAG adds 1,027 g/mol to the protein.6)What types of material can be conjugated?MSD GOLD SULFO-TAG NHS-Ester is reactive with primary amines. Proteins and large peptides are easily labeled. Fab fragments have also been conjugated successfully.MSD Conjugation Services may be used to conjugate small molecules and peptides. Please contact MSD Scientific Support (Phone: 1-240-314-2798, Email: ScientificSupport@) or your local MSD Application Scientist for details.7)What conjugation ratio is recommended for an IgM?A challenge ratio range from 8:1 to 12:1 may be used for conjugating IgM antibodies. The molecular weight of an IgM is inthe order of 900,000 Da. IgMs can be unstable at higher pHs, therefore conjugation at pH 7.0 to 7.2 may be better than the standard labeling buffer of pH 7.9 used for IgG.8)Are there alternatives to using Conjugation Buffer for the conjugation reaction?For best results, it is recommended to use Conjugation Buffer. However, other buffers can be used for the conjugation reaction provided they are free of amine-containing molecules (i.e., no Tris- or glycine-containing buffers) and preservatives. Affinity-purified antibodies are commonly eluted with high molarity glycine solutions; therefore, it is important that they are properly desalted prior to conjugation. Using alternative conjugation buffers may result in lower incorporation efficiencies.9)What should I do if my application requires the conjugated protein to be in a different buffer?The desalting columns may be equilibrated in a buffer other than PBS if the end application requires storage of the conjugated protein in a non-PBS buffer.10)Will my antibody retain activity after labeling?MSD GOLD SULFO-TAG is a small hydrophilic molecule and generally does not affect the function of its conjugation partner, especially when labeling large proteins such as antibodies. With small molecule or peptide labeling, the addition of MSD GOLD SULFO-TAG may have an effect on binding affinities.11)What is the stability of MSD GOLD SULFO-TAG NHS-Ester?The shelf life of MSD GOLD SULFO-TAG NHS-Ester is 3 years at ≤-70°C. The reagent can be stored for up to 2 years at ≤-10°C with minimal loss of activity. Reagent stability is lower at room temperature or at 2–8°C. At room temperature, there may be a 1/3 to 1/2 loss of active material in a month. Once the reagent is reconstituted, it should be used as soon as possible since the NHS-ester hydrolyzes in water. After reconstitution, the solution may be kept up to 10 minutes on ice with minimal loss of activity.12)What if the protein to be conjugated does not have any primary amine groups?Alternative linking chemistry options are available from MSD which allow non-amine containing molecules to be successfully labeled. These include Thiol-Reactive linker (SULFO-TAG Iodoacetamide), Carboxyl (-COOH) Reactive linker (SULFO-TAG Amine) and Carbohydrate Reactive SULFO-TAG. Please contact MSD Scientific Support (Phone: 1-240-314-2798, Email: ScientificSupport@) or your local MSD Application Scientist for details.13)I have an IgG purified antibody from my protein production group which has been eluted into PBS. Should Idesalt before conjugation?Yes. Tris-glycine is a major component of antibody elution buffers used in the purification procedure. On many occasions,a single desalting into PBS is insufficient. It is recommended to repeat the desalting step into PBS to remove any tracequantities of Tris-glycine that can hinder conjugation with MSD GOLD SULFO-TAG.14)How do I conjugate a high concentration protein solution with MSD GOLD SULFO-TAG?The conjugation reaction will be more efficient at high protein concentrations. It is recommended to use a lower challenge ratio for conjugation to compensate for the increased efficiency.15)How do I conjugate a low concentration protein solution with MSD GOLD SULFO-TAG?MSD recommends the protein concentration to be at least 0.5 mg/mL. If concentrating the protein solution is not feasible, conjugation can be done at a lower concentration, which may result in lower conjugation efficiency. Therefore, the conjugation reaction should be performed at a challenge ratio of 20:1 or higher.16)How do I conjugate small proteins with MSD GOLD SULFO-TAG?Proteins with MW < 40,000 Da can be conjugated by the same chemistry as antibodies; lower challenge ratios may be required for MSD GOLD SULFO-TAG conjugation of small proteins and peptides than for IgGs. The NHS-Ester will react with primary amines such as lysine residues and the N-terminus of proteins and peptides. If there is no primary amine available, a different chemistry will be necessary. Post-conjugation purification of small proteins will require alternative procedures (such as HPLC, FPLC, etc.) because small proteins are not resolved by G-50 columns.17)Why can't I use a spectrophotometer at 280 nm to determine conjugated protein concentration?MSD GOLD SULFO-TAG strongly absorbs at 280 nm and will interfere with any measurement of protein concentration at this wavelength.18)What is the stability of MSD GOLD SULFO-TAG conjugated proteins?MSD GOLD SULFO-TAG conjugated protein is generally as stable as the unconjugated protein if it is stored in the appropriate buffer, concentration, and storage temperature. The conjugated protein should be stored in the dark, either at 2–8°C or frozen in aliquots. Azide should be added for long term storage at 2–8°C to prevent any microbial growth. If the protein concentration is low, consider adding a carrier protein, such as 0.1% MSD Blocker A.19)My antibody did not conjugate very well. What are the possible reasons?The presence of preservatives, carrier protein or residual Tris-glycine or other interfering substances in the conjugation buffer (see FAQ 1 and 2) can reduce conjugation efficiency of the protein. Very low concentrations of the starting material (below 0.5 mg/mL) may also reduce conjugation ratios. It has also been observed that some IgGs label more efficiently than others.20)What components can be removed by buffer exchange or dialysis?Salt, azide, glycerol, buffering agent (e.g., Tris), carbohydrates (e.g., trehalose), and amino acids (e.g., histidine, glycine) can be successfully removed by buffer exchange method.21)Who should I contact if I have any questions on MSD GOLD SULFO-TAG conjugation?Please contact MSD Scientific Support (Phone: 1-240-314-2798, Email: ScientificSupport@) or your local MSD Application Scientist for details.WorksheetDate:MaterialsProtein to be conjugatedConcentration: Vendor:Catalog number: Lot number:Sample PreparationMethod: Buffer:Lot number: Date:Columns/Concentrators: Lot number:MSD GOLD SULFO-TAG NHS-Ester ReconstitutionSize: Lot number:Distilled water:Lot number: Date:Volume of water added to vial: Stock concentration (nmol/µL):Separation and CalculationsBuffer:Lot number: Date:Columns: Lot number:Protein assay kit:Type: Lot number:Pre-Conjugation Calculations1,000 x Protein conc. (mg/mL) x Challenge ratio x Volume of protein solution (µL) = nmol of SULFO-TAG reagent required Protein MW (Da)nmol of SULFO-TAG reagent required = µL of SULFO-TAG stock solution required for conjugation reaction Conc. of SULFO-TAG stock solution (nmol/µL)。

Cellular Thermoshift Assay Protocol(Adapted from Nature Protocols Vol.9, No.9, 2014)Part 1: Determination of melting curve for intracellular protocol-- Protocol for Suspension Cells1.Grow Jurkat cells to cell density of 2e6 per ml2.Add 15 ml of cells at 2e6/ml density into T75 flasks3.Add 30ul of 10mM DMSO stock solutions of compounds to 20uM final concentrations (onecompound per flask). Use similar volume of DMSO for control.4.Incubate cells 1h with compound at 37 degrees in TC incubator5.Collect cells into 15 ml tube6.Centrifuge tubes 300xg for 3 min to pellet then remove medium7.Wash cells: Resuspend cells in 15 ml of PBS and then centrifuge 300xg for 3 min to pellet andremove medium8.Add 1 ml of PBS with protease inhibitors to each cell pellet9.Distribute cells 100ul from each 15 ml tube to 10 different 0.2ml PCR tubesing gradient thermocycler heat 6 tubes of the cell suspensions between 40-55 degrees for 3minutes (make sure thermocycler is at desired temperature before adding tubes).11.Remove tubes and place at room temperature for 3 minutes, then snap freeze in liquid nitrogen12.For the remaining four tubes follow similar procedure but heat between 58-67 degrees.13.Samples can be stored for future analysis at this step14.Lyse cells by freeze-thaw 2 times between liquid nitrogen and a 25 degree heat block. Vortex ateach freeze-thaw cycle and store at 4 degrees after final thaw.15.Centrifuge cell-lysate containing tubes at 20,000 x g for 20 min at 4 degrees to pellet cell debrisas well as precipitated and aggregated proteins16.Transfer 90ul from tube without disturbing the pellet to a new tube.17.Run western blot using 40ul of lysate mixed with 20 ul of reducing loading buffer. Of thismixture it is advised to load 13ul on a gel for western detection.18.Quantitate band intensity and plot with temperature curve.Part 2: Determination of Dose-response fingerprint for each compound for an intracellular protein—Protocol for suspension cells1.Choose temperature established in Part 1 in which most of the protein is precipitated and notdetected in the absence of stabilizing compound (eg. DMSO control) but at which a majority of protein remains soluble in the presence of saturating compound concentration (20um).2.Similar to Part 1, grow cells to 2e6 cells per ml3.Collect cells to a 15 ml and pellet at 300 xg for 3 min4.Resuspend cell pellet in culture medium to density of 40 million cells per ml5.Make 11 point dose curve-- Make dilution of compounds in a round bottom 96 well plate. Place15 ul of 4 mM DMSO stock solutions of different compounds in a 96 well plate in Row 1. Add10ul of DMSO into remaining row. Make serial dilutions by transferring 5ul of 4mM compound from well A down the row to make serial dilution6.Place 5 ul of into new 96 well plate and dilute 50 fold in culture medium7.Transfer 50 ul of diluted compound to PCR tubes or 96 well PCR plate8.Add 150 ul of cell suspension from #49.Incubate tubes for 30 min at 37 degrees shaking tube or plate every 10 minutes10.Heat the tubes or plate in a pre-heated thermocycler for 3 min at temperature determined from#1.11.Cool Plate for 3 min at RT (Use a room temperature heat block if possible)12.Transfer 50ul of samples to PCR strips for freezing and snap freeze in liquid nitrogen13.Samples can be stored here at -80 degrees14.Freeze-thaw cells twice using liquid nitrogen and a 25 degree heat block. Vortex at each thawstep15.Spin tubes at 20,000xg for 20 min at 4 degrees to pellet cell debris and precipitated protein 19.Remove 40 ul from tubes and mix with 20 ul of reducing loading buffer. Of this mixture it isadvised to load 13ul on a gel for western detection.20.Quantitate band intensity and plot with temperature curve.。