I can see the protein band on blot after staining with Ponceau. but at the time of developing the blot. there is no result but staining the blot with Ponceau show bands clearly. I have changed PBST (0.5%), primary Ab, Sec. Ab, developer and fixer as well as ECL.
How many times a blot can be used to develop with & without stripping.
Do i have to run gel again with the same sample.
Shradha,
It sounds like you have changed out all of the likely issues. I don't usually strip and reprobe a blot more than 4 times. If you have tried to probe for your sample more than that, I would suggest re-running the gel with ideally fresh sample.
Do you have a positive control for the protein that you are trying to detect that you can run side-by-side with your sample? Perhaps the primary antibody you are using isn't compatible with Western blotting. If a suitable positive control is not available to confirm your primary antibody, I would try to order a different primary for your protein. This sounds like a mismatch between antibody and antigen.
Hope this helps and good luck.
You may need to optimize conditions for detection of your protein using different amounts of protein and different dilutions of the primary and secondary antibody. As noted by Chris, I would include a positive and a negative control.
I suggest you to expose the gel for 2 hours.. please also choose a good HRP detection kit, it makes a great difference..
Check for the suitable antibody dilution and temprature for incubation.. Also do not use too old antibodies if you have antibodies that are like 2-3 year old use biotin with your primary and secondary antibodies.
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