Rinse the blot in TBST three to four times for 5 min each at room temperature.We recommend incubating in a sealed bag, hybridization tube or 50 ml Falcon tubes (~2.5 ml primary antibody/blot). Dilute the primary antibody in TBST to the recommended dilution.Block the membrane with 5% w/v BSA in TBST.We would recommend not washing blots in distilled water as this can strip off proteins in some circumstances. The stain can be removed by washing in PBST or TBST. If required, the efficiency of transfer can be determined by staining the membrane briefly (10 sec) in Ponceau stain.Please note: for PVDF it is essential to pre-wet the membrane in methanol prior to transfer. Transfer the proteins to a PVDF membrane using semi-dry or wet transfer methods.Load the sample onto an SDS-polyacrylamide gel and run the gel under standard conditions.Denature the proteins by heating the sample to 95☌, or boiling, for 5 min.Seize one look at our BETA site and see whatever we’ve done so far. Were improving press wed welcome your feedback. Were improving and wed welcome your reaction. For non-reduced samples, the DTT or ß-mercaptoethanol is not added. Include a list of reagents and a step-by-step procedure. Proteins come up as clear zones in a translucent blue background. Wash the gels briefly in de-ionized water, and view them against a dark-field background. For reduced samples, the sample buffer should be supplemented with DTT or ß-mercaptoethanol. Briefly rinse freshly-electrophoresed gels in distilled water (30 sec maximum) and then transfer to a solution of 0.3 M CuCl 2 for 515 min. To a sample of protein solution containing 1-100 ng of the target protein (500 µg lysate), add an equal volume of 2x SDS-PAGE sample buffer.Remember to add phosphatase inhibitors to cocktails bought when investigating phosphorylation events.
Use a RIPA or NP40 buffer supplemented with fresh protease and phosphatase inhibitor cocktails. 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. Homogenize the cells or tissue of interest in lysis buffer made fresh and containing a cocktail of protease inhibitors (and phosphatase inhibitors when dealing with phosphorylated proteins).Īs soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. Procedure for detection of phosphorylated proteins.
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