western blotting

Western blottingIntroductionThe electrophoretic transfer of proteins from sodium dodecyl sulphate polyacrylamide gels (SDS-PAGE) to sheets of nitrocellulose was initially referred to as “Western” blotting. In order to avoid this geographical jargon, the term immunoblotting is used.Principles●Western blotting: It is an experimental method in immunology. We transfer the proteinsand fixed them to a man-made membrane. Then we use the same method as in ELISA, inwhich the first antibody and the second one are added in turn. Then the enzyme labeledsecond antibody will react to cause a change of color, when the substrates are added. In thisway we can reveal the result in the membrane to show the marks. We can see the resultdirectly just by our eyes.●Immobilization: To transfer the proteins to the membrane, we use SDS-PAGE as theintermediate method. This method helps to separate the different proteins into differentbands and purify them. We use the blotting tank as the equipment in the step ofimmobiliza tion. In this way, the proteins’ bands are replicated to the fibrin membrane,which will be the reactive place for the following reactions.●Immunoblotting: When the enzyme bound to the second antibody react with the substrates ,the band in the membrane will be visualized by this reaction which cause the color change.ReagenstsTable 1 ReagentsProcedureTable 2 Experimental procedureTime Experimental procedure Phenomenon8:30-9:30 9:30-11:0011:20-13:10 SDS-PAGE：Preparation: wash the plate and draw mark.Prepare SDS-PAGE Gels to run samples on it.Load samples and run the gel in the voltage of100VWe made two 7.5% running gel.In this process, we have failed to make thesecond gel several times due to the airbubble between two plates. At last, we pressthe bubble away and run the next step.We design the sample loading as follows:13:10-13:251)Take the gels from gel box, then cut GEL 1 into two separate gels between well No.5 and No.6.Stain the left half of GEL 1 (well No.1-5) with Coomassie Blue overnight. Distain it later to make the original copy to compare with the other half of the gel which is for transferring.The band of bromophenol blue is 1.2cm away form the bottom of the gelThe Coomassie Blue is not fresh and the staining result is not satisfactory.13:30-15:40Transfer:Prepare two pieces of nitrocellulose membranes each in exactly the same size as the right half (well No.6-10) of GEL 1 and GEL 2. Prepare 8 copies of filter paper for each gel in the same size. Soak the membrane, filter paper and the gels in the transfer buffer for at least 15min, be sure to drive off the bubbles between them.Place the gel, paper, and membrane into the blotting tank in the correct order. 4 sheets filter papers membrane Gel4 sheets filter papersClose the tank, then make constant-current transferring electrophoresis for 1.5h. The value of the current is calculated form the area of the gels.1)We use the gloves to avoid contaminating the nitrocellulose membrane.It isn’t very easy to get these pieces so tidy . We spend a short period of time in doing so.According to the formula (I =Gel surface area A /cm 2×0.8mA), we get the exactly value of Transfer time: 14:10-15:40, during this time PBS-Tween-20 solution is prepared.overnightnext day: 8:30-10:3010:30-11:1511:15-12:45Immuno-detection:1) The membrane is soaked in block solution to block non-specific protein-binding sites at 4℃ overnight.2) wash the membranes with fresh PBS-Tween-20 and keep in this environment3) cover the membranes with the first antibody solution into the plastic bags for 2h.4) wash the membranes with fresh PBS-Tween 20 for 3-5min to remove unbinding antibodies.5) Cover the membranes in the second antibody solution into the plastic bags, shake the plastic bags for 1.5h.6) Wash the membrane with PBS-Tween-20 3-5min.7) Develop the membranes in 10ml substrate solution.8) Stop the developing process in ddH 2O and dry the membranes in filter paper directly.9) Dry the blot on filter paper and store in the notebook.The membrane 2 is broken, but not due to our fault.Membrane 1: in 5ml blocking solution; Membrane 2: in 10ml blocking solution.We chose Rabbit-anti-Human IgG antiserum as the first antibody for Membrane 1.Dealing with Membrane 2, we cut it into two halves: the left half is for negative control (adding no first antibody), the right one is added with Rabbit-anti-Horse IgG antiserum as the first antibody.The half of membrane 1 and both halves of membrane 2 are soaked.We chose Goat-anti-Rabbit IgG-HRP as the second antibody.Each washing step in the PBS-Tween-20 is very important, for it can affect the coloration of membranes.Antigen bands can be visualized suddenly, so we should do this process quickly.The bands on the western blotting are good, and the negative control has no band on it, as supposed.Up + Down-ResultFig 1. the compare of SDS and western blotting in Sample I**This is the western blotting(right) compared with the SDS-PAGE(left).(1) The four bands of well No.2 stand for the standard protein, which in fact contains fiveproteins. However, the concentration of running gel is only 7.5% rather than 12.5%, which might cause one band of the proteins disappeared. The concrete analyze will be seen in the discussion later.(2) The bands of well No.3,4,7,8,9 stands for Human IgG(5μl), Horse IgG(10μl), HumanIgG (5μl), Human IgG (10μl) and Horse IgG(5μl),repectively.(3) Since the first antibody of this sample is Rabbit-anti-Human IgG antiserum, thebands of well No.7 and No.8 in the membrane is much darker and thicker than those of well No.9, as it was supposed.Fig 2. The western blotting in Sample 2, compared with negative control(1) The figure on the left is the left half of the membrane 2 which was used for control. In thiscontrol the first antibody wasn ’t added. In fact the samples loaded into well No.2,3 and 4 are std, protein(5μl), horse IgG(5μl) and Human IgG(10μl) respectively.(2) The bands of well No.7,8,9 stands for Horse IgG(5μl), Horse IgG(10μl), Human IgG(5μl),respectively.(3) Because the first antibody of this sample is Rabbit-anti-Horse IgG antiserum, thebands of well No.7 and No.8 in the membrane is darker and thicker than those of well No.9, wholly opposed to sample 1.Fig 3. The western blotting in Sample 2, compared with the gel which had beenused to transfer.(1) The gel on the left is exactly the gel used to transfer to the membrane on the right.(2) The bands of the two pictures fit each other for the most part, but there is some slight bandshowed on the membrane rather than on the gel.1 2 3 4 5 6 7 8 910 Human IgG(5μl) Horse IgG(10μl) Horse IgG(5μl) Human IgG(10μl) Human IgG(5μl) Std. MW(10μl)Human IgG(5μl) Horse IgG(10μl) Horse IgG(5μl)12345 678910Human IgG(5μl) Horse IgG(10μl) Horse IgG(5μl)Discussion● About the standard protein:As told, the standard sample we use for mark has five protein Phosphorylase b, BSA,Carbonic anhydrase, Ovalbumin, Lysozyme.Unexpectedly, there are only four bandsappearing after destaining. The concentration ofrunning gel is 7.5%, size range of molecules forthis concentration is 45 000-200 000, it ’s likelythat two of the smallest standardprotein-Carbonic anhydrase(M r=30 000) andLysozyme(M r=14 300) can ’t be separated andbecome overlapped(fig. 4).Fig. 4 Protein standard ● About the first antibody:In this experiment, we used both of the first antibody: Rabbit-anti-Human IgG antiserum and Rabbit-anti-Horse IgG antiserum. Seeing the membrane, we can ’t easily find that both antibodies have the affinity of binding Human IgG and Horse IgG . That mean cross-binding was happening. We can conclude that there is some similar segments in both antigen. These uniform segments are able to bind the same antibody. But conspicuously, the affinity of cross-binding is smaller than that of real binding. Apart from this, we can also find that the ability of rabbit-anti-human antiserum to bind horse IgG is stronger than that of rabbit-anti-horse antiserum to human IgG , for the No.9 band of membrane 1 is clearer than that of membrane 2. Maybe it does show some evidence of structural distinction between human IgG and Horse IgG .● About the tailing banding.Seeing the figure on the right, we can the many unexpected banding. There must be some other proteins which was too few to be colorated by Coomassie Blue. Yet it does have the affinity to bind the antiserum. Maybe the plausible explanation is that: the antigen is not pure, and the first antibody also have the affinity to bind the impurity. Hence the tailing band appeared. But it suggests that the western blotting is so sensitive that it can show the protein unable to be showed by only staining.Fig. 5 Tailing banding● About the negative controlIn our experiment, we did a control in which no first antibody is added, just like the ELISA.After dipped in substrate, it appears no band on it. But it ’s not enough. We should have done more controls, such as a control without second-antibody, a control without blocking etc. But I think it is enough to verify that the special affinity of first antibody to bind the antigen.● About the affinity and specificity of binding.We know that SDS will destruct the tertiary structure of proteins. It seems that the antigen handled by SDS will lose the affinity to bind the antibody. But it didn ’t happen. The SDS should have not wholly destroyed the affinity of antigen to bind antibody. It shows the strong affinity and specificity of binding.● About the transferring.Compared two the picture of Fig.3, we can find that the protein on gel wasn ’t wholly transferred to the membrane. But it ’s enough to show the bandclearly on themembrane. Maybe it is possible to transfer the protein from one gel to more than one membrane. Then we can try different antibodies for these membranes.Reference:Bingbin YU. The Technique of Experimental Biochemistry. Tsinghua University, 2001Phosphorylase b M r=97 400 BSA Mr=66 000 Ovalbumin M r=43 000 Carbonic anhydrase and Lysozyme (overlapped)Tailing band。