Otherwise, the antibodies or other detection reagents will bind to any remaining sites on the membrane that initially served to immobilize the proteins of interest. In principle, any protein that does not have binding affinity for the target or probe components in the assay can be used for blocking.
A variety of blocking buffers ranging from milk or normal serum to highly purified proteins can be used to block free sites on a membrane. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether without altering or obscuring the epitope for antibody binding.
Blocking buffers can influence antibody binding and specificity- so optimization is needed. No single protein or mixture of proteins works best for all western blot experiments, and empirical testing is necessary to obtain the best possible results for a given combination of specific antibodies, membrane type and substrate system.
Washing steps are necessary through the series of incubations to remove unbound reagents and reduce background. A variety of buffers may be used. Detergents such as Tween can be added to the buffer to help remove nonspecifically bound material. The amount of Tween 0. Weak binding antibodies may be washed away by too much detergent. In applications where alkaline phosphatase conjugates are used, TBS should be selected as PBS interferes with alkaline phosphatase.
When performing fluorescent western blotting, it is recommended to eliminate the detergent from the blocking step. Detergents auto-fluoresce and will contribute to higher background. Reprobing a western blot saves time and conserves sample while allowing optimization to be performed as needed. These specially formulated stripping buffers are designed to dissociate and strip primary and secondary antibodies from western blots so that membranes can be reprobed under alternate conditions or with another antibody to detect a different protein target.
Solution : Membrane treatment reagent and a primary antibody diluent that increases both signal intensity and sensitivity 3- to fold compared to a detection performed without it. Need : Obstructed detection of target protein due to interference from both heavy-chain and light-chain IgG fragments from immunoprecipitation assay.
Solution : HRP conjugate that is optimized for post-immunoprecipitation western blot detection of primary antibodies without interference from denatured immunoprecipitation IP antibody fragments. Download Technical Tip: Stripping and reprobing western blots.
Western blot protocols and buffer recipes. Download: Western Detection Workflow Brochure. Explore: Protein Gels. Don't have an account? Create Account. Sign in Quick Order. Search Thermo Fisher Scientific. Western blot sample prep. Transfer and staining. Membrane stripping. Troubleshooting tips. Western blot FAQ. Western blot products. Loading control guide. Optimized secondary antibodies for western blotting.
Other protocols. View all protocols. Video protocols library. RIPA buffer radioimmunoprecipitation assay buffer RIPA buffer contains the ionic detergent sodium deoxycholate as an active constituent and is particularly useful for nuclear membrane disruption for nuclear extracts.
A RIPA buffer gives low background but can denature kinases. Load samples. Run the gels at V until the dye front reaches the bottom of the gel approximately 60 minutes.
Soak nitrocellulose membrane and blotting paper in 1X transfer buffer for at least 5 minutes prior to opening gel cassette. Open gel cassettes and place the gel on the nitrocellulose membrane sandwiched between two pieces of blotting paper. Place in transfer apparatus and fill with fresh 1X transfer buffer.
Run transfer apparatus for minutes on 35V. For best results, the optimal dilution of antibody should be empirically defined. Incubate the membrane in diluted primary antibody for two hours to overnight with gentle shaking at room temperature.
Wash the membrane three times, 10 minutes each time in TBST.
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