I have attached a picture for my most recent western blot. My Beta-actin always shows up completely perfect but all other protein blots appear gray and messy. This has happened multiple times. What can I do to fix this? I know it is not due to faulty antibodies or buffers since everyone else has been getting normal results.
I have a few questions that might help me understand your issue more clearly
1. Are you using nitrocellulose or PVDF
2. Are you using chemiluminscence of FL detection
3. What blocking reagent are you using
4. Is the protein that you are looking at abundant or in low quantity
5. How long are you washing your blots between antibodies and with what percentage of tween 20.
It is difficult to assess without knowing the above. I know that you said that your coworkers are using the same reagents but maybe they are doing a step that is second nature to them that they do not realize they are even doing and forgot to tell you. Have you observed them perform the entire process?
hi
1- you should increasing blocking time (overnight at 4°C with gentle shaking )
"Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA or 5% nonfat dry milk as indicated on primary antibody datasheet; for 20 ml, add 1.0 g BSA or nonfat dry milk to 20 ml 1X TBST and mix well."
2- Wash well between the steps (3X10 min)
3- in detection step >> Drain membrane of excess developing solution (do not let dry), wrap in plastic wrap and expose to x-ray film. An initial 10 sec exposure should indicate the proper exposure time.
https://www.cellsignal.com/contents/resources-protocols/western-blotting-protocol/western
good luck
In addition to the above comment you may have trouble with transfer of your proteins. Beta actin is a protein of relatively low molecular weight and higher molecular weight proteins may heve transfered with less efficacy. You would be sure of the transfer efficacy with a Ponceau staining. Is it normal?
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