![]() ![]() Please see the suggestions below for tips on optimizing your WB on phosphorylated proteins.ġ. ![]() After washing with tap water to remove residual fixative, dry at room temperature.Ĭarrying out western blots on phosphorylated proteins can require further optimization and a few unique conditions. After development, the film should be immediately immersed in the fixing solution to a transparent film. Take out the film at a red light, open the film clip, put the film on the membrane, remove the film clip, adjust the exposure time based on the strength of the signal, remember that an over-exposed film is not suitable for analysis.Īfter the exposure is complete, remove the film, quickly immerse in developing solution, terminate development. Ensure the membrane surface proteins have sufficient contact with the mixture. In darkroom, load the developer and fixative respectively into plastic trays mixing the two reagents, volume of A and B, in a centrifuge tube. Incubate with the secondary antibody, the same as step (4), dilution with TBST, 37℃ shaking incubation 1 ~ 3 h.Ĭhemiluminescence, developing and fixing, for HRP-conjugated antibodies: ECL are the traditional kits used. Then load the membrane with a ziplock bag and fixed lamination machine, add the antibody solution to the ziplock, and make sure the membranes are incubated with sufficient volume of antibody.ĥ) 37℃ shaking incubation 1 ~ 3 h (according to the antibody titer and affinity ), or 4 ℃ overnight incubation, TBST wash on the shaker at room temperature for 3 times, each time 5 ~ 10 min.Ħ). According to the recommended antibody dilution, dilute antibody with TBST (or 1% to 3% non-fat milk). Blocking at 4 ℃ with shaker shaking overnight, or 37 ℃ blocking for 2 hours. Block the membrane generally with 5% non-fat milk, or 3%~5% BSA. Then rinse off the dye with distilled water.ģ). After the transfer, use Ponceau dye staining about 2 ~ 5min to check the transfer is successful or not. ![]() (according to the instructions recommended by trans-blot system.)Ģ). Transfer the protein to the membrane from the gel. Transfer of proteins and staining (Western blotting)ġ). Subject to electrophoresis according to the instructions recommended by electrophoresis method, Terminate the gel electrophoresis when bromophenol blue runs out the gel. Load samples and run the gel, each hole sample volume 20-40μg protein, pay attention when spotting the sample, do not overflow into adjacent wells which will cause gum poked holes and cross-contamination.ĥ). 4% Stacking gel, select pre-stained protein Marker Proteins in size, to help distinguish the size and protein electrophoresis tracer.Ĥ). Prepare PAGE gels, choose different concentrations of separating gel depending on the molecular weight of the protein:ģ). Clean the glass and set up the electrophoresis system.Ģ). To denature, use a loading buffer with the anionic denaturing detergent sodium dodecyl sulfate (SDS), and boil the mixture at 95-100☌ for 5 minutes.ġ). Preparation of samples for loading into gels Bovine serum albumin (BSA) is a frequently-used protein standard. Use PAGE gels, a Bradford assay, a Lowry assay or a BCA assay. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice discard the pellet. The RIPA (with 1mM pmsf ) should be ice-cold prior to homogenization.Ĭut the tissue into small pieces, For a ~20 mg piece of tissue, add ~250 μl lysis buffer rapidly to the tube, homogenize with an electric homogenizer (or a glass homogenizer) until fully lysed.Ĭentrifuge for 5 min at 10000~14000g at 4☌ in a microcentrifuge. Pmsf (Phenylmethanesulfonyl fluoride) is the protease inhibitors we always use.ĭissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases. These events can be slowed down tremendously if samples are kept on ice or at 4☌ at all times and appropriate inhibitors are added fresh to the lysis buffer. ![]() We use RIPA buffer (beyotime P0013B) for whole cell extracts and membrane-bound proteins.Īs soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. This solubilizes the proteins so they can migrate individually through a separating gel. To prepare samples for running on a gel, cells and tissues need to be lysed to release the proteins of interest. ![]()
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