I am doing western of mice organ extracts. I am using 3% BSA and overnight blocking followed by 6 hour primary antibody incubation at 4 degree and then 2 hour secondary antibody incubation.
The problem i am facing too much of non-specific bands and more backgrounds in my blot?
i try to standarize the conditions even then i got the same results.
What i do to overcome this ?
Thank you in advance!
Hello Himanshu ...
1. Poor handling of the sample prior to Western Blot can result in multiple bands due to degradation of the sample.
2. You could load 20-30ug total protein per well. Avoid loading more protein unless and until the protein of interest is in low abundance. If excessive amount of lysate is loaded on the gel, antibody binds non-specifically to proteins which is in excess resulting in multiple bands.
3. You could use 5% BSA in TBS-T (TBS with Tween 20) for blocking for 1-1.5 hours.
4. Use primary antibody in the range of 1:2000 to 1:5000 dilution and incubate with primary antibody overnight at 4 degree C. Use of high concentration of primary antibody could lead to non-specific bands. Prepare primary antibody in 3% BSA in TBS-T.
5. Use secondary antibody in the range of 1:10000 to 1:20000 dilution and incubate with secondary antibody for 1 hour at room temperature. Prepare secondary antibody in 3% BSA in TBS-T. Avoid secondary antibody at high concentrations.
6. Use good quality antibodies because contaminated or unpurified antibodies will result in multiple bands.
7. Sometimes, if the protein is cleaved post-translationally and if you are using polyclonal antibody, then multiple bands could be observed because of the ability of polyclonal antibody to recognize multiple epitopes.
8. The washing steps should be performed 3 times (5mins each) or may be more.
9. The incubation and washing steps must be performed on a low-speed shaker.
10. Lastly, do not allow the membrane to dry during the Western Blot procedure.
You may modify the above points as per your requirements to obtain better results.
Good Luck.
If your gel as well as ponceau staining after transfer on the membrane show the good banding pattern, then the issue of protein degradation can be ignored. Once it it confirmed then you must look at other aspects of WB procedure.
The generation of nonspecific bands with more background is mainly due to the use of blocking buffer or dilution/overexposure of antibodies (primary/secondary antibody).
You can replace the blocking buffer by nonfat milk or Nonidet p-40 (non-ionic detergent) in order to minimize the background noise.
You can make a brief modification in antibodies dilution but try to follow the company manual protocol in order to minimize the high background and enhance the signal of protein of interest.
I would suggest you to check once again the company datasheet of the both antibodies (primary as well as secondary) and accordingly follow the dilution as well as exposure time.
Hope you might be following other basic procedural aspects of WB carefully in order to minimize other possibilities that are associated with the generation of nonspecific bands.
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