【资源】Western blot技巧，新鲜出炉!
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你注意过以下技巧吗?
1. Milk-based blockers may contain IgG that can cross-react with anti-goat antibodies. This can significantly increase background and reduce antibody titer. Milk may also contain endogenous biotin or phospho- epitopes that can cause higher background.
2.To stretch the Odyssey Blocking Buffer: recycle your blocking buffer for antibody dilution; dilute 1:1 in PBS; save excess used blocking buffer at 4°C for several days for re-use (use a separate container – do not return to the main bottle).
3.Store the antibody vial at 4°C in the dark. Do not thaw and refreeze the vial, as this will affect antibody performance. Minimize exposure to light and take care not tointroduce contamination into the vial. Dilute immediately prior to use. If particulates are seen in the antibody solution, centrifuge before use.
4. Protect membrane from light during secondary antibody incubations and washes.
5.Use the narrowest well size possible for your loading volume to concentrate the target protein.
6.The best transfer conditions, membrane, and blocking agent for your experiments will vary, depending on the antigen and antibody. If you have problems with high back ground or low signal level, a good first step is to try a different blocking solution.
7.For maximum sensitivity, use nitrocellulose membrane for transfer.
8.Small amounts of purified protein may not transfer well. Adding non-specific proteins of similar molecular weight can have a “carrier” effect and substantially increase transfer efficiency.
9.For proteins <100 kDa, try blotting in standard Tris-glycine buffer with 20% methanol and no SDS. Addition of SDS to the transfer buffer can greatly reduce binding of transferred proteins to the membrane (for both PVDF and nitrocellulose).
10.Soak the gel in transfer buffer for 10-20 minutes before setting up the transfer. Soaking equilibrates the gel and removes SDS so that it will not be carried over into the transfer tank.
11.To maximize retention of transferred proteins on the membrane, allow the membrane to air-dry completely after transfer (approximately 1-2 hours).
12. Do not over-block. Long blocking incubations, particularly with nonfat dry milk at 2% or higher, can cause loss of target protein from the membrane (J. Immunol. Meth. 122:129-135, 1989).
13.To enhance signal, try extended primary antibody incubation at room temperature or overnight incubation at 4°C. Avoid extended incubations in secondary antibody.
更多技巧及western blot问题解答，例如如何降低wetern blot的背景等，可参考如下文献:
http://biosupport.licor.com/docs/whatsnew/Western_Proto_09288.pdf
1. Milk-based blockers may contain IgG that can cross-react with anti-goat antibodies. This can significantly increase background and reduce antibody titer. Milk may also contain endogenous biotin or phospho- epitopes that can cause higher background.
2.To stretch the Odyssey Blocking Buffer: recycle your blocking buffer for antibody dilution; dilute 1:1 in PBS; save excess used blocking buffer at 4°C for several days for re-use (use a separate container – do not return to the main bottle).
3.Store the antibody vial at 4°C in the dark. Do not thaw and refreeze the vial, as this will affect antibody performance. Minimize exposure to light and take care not tointroduce contamination into the vial. Dilute immediately prior to use. If particulates are seen in the antibody solution, centrifuge before use.
4. Protect membrane from light during secondary antibody incubations and washes.
5.Use the narrowest well size possible for your loading volume to concentrate the target protein.
6.The best transfer conditions, membrane, and blocking agent for your experiments will vary, depending on the antigen and antibody. If you have problems with high back ground or low signal level, a good first step is to try a different blocking solution.
7.For maximum sensitivity, use nitrocellulose membrane for transfer.
8.Small amounts of purified protein may not transfer well. Adding non-specific proteins of similar molecular weight can have a “carrier” effect and substantially increase transfer efficiency.
9.For proteins <100 kDa, try blotting in standard Tris-glycine buffer with 20% methanol and no SDS. Addition of SDS to the transfer buffer can greatly reduce binding of transferred proteins to the membrane (for both PVDF and nitrocellulose).
10.Soak the gel in transfer buffer for 10-20 minutes before setting up the transfer. Soaking equilibrates the gel and removes SDS so that it will not be carried over into the transfer tank.
11.To maximize retention of transferred proteins on the membrane, allow the membrane to air-dry completely after transfer (approximately 1-2 hours).
12. Do not over-block. Long blocking incubations, particularly with nonfat dry milk at 2% or higher, can cause loss of target protein from the membrane (J. Immunol. Meth. 122:129-135, 1989).
13.To enhance signal, try extended primary antibody incubation at room temperature or overnight incubation at 4°C. Avoid extended incubations in secondary antibody.
更多技巧及western blot问题解答，例如如何降低wetern blot的背景等，可参考如下文献:
http://biosupport.licor.com/docs/whatsnew/Western_Proto_09288.pdf
