My colleague suggested me that in order to check that antibody is degraded or not, run that antibody for a western and check for the release of bands. If the antibody is degraded then i might find two bands in a gel. is it so?
Secondly even if i want to run that particular primary antibody what amount of antibody i will have to load in to a well?
If you want to check your antibody by Western Blot you have to take care about the conditions:
-no beta-Mercaptoethanol in the loading buffer
-you don't have to perform WesternBlot, you can also to a Coomassie staining
- for Coomassie staining about 0,5 µg are ok, for WesternBlot take less (0,1 µg)
Degradation means, you will have two bands for the antibody: around 55 kDa and 25 kDa
Assuming you are doing Western blotting, two band in the gel might result from cleavage/degredation of the target protein of interest, not your primary. Not to mention non-specific binding. For example, B-actin can cleave into two fragments under certain circumstances.
Primary antibody concentration needs to be optimized, try 1:200, 1:500, 1:1000 and 1:2000.
If you are doing Western/immunoprecipitation, the degradation of Ab will be like you will not get the signal with the secondary. Degradation of primary will either not bind with the motif or will bind with motif but will not be able to ligand with the secondary, as mostly the degraded primary will wash off during wash.
It is really an unique question, but i think repeated no signal yield can only suggest you of such degradation.
Best,
Somnath.
If you want to check your antibody by Western Blot you have to take care about the conditions:
-no beta-Mercaptoethanol in the loading buffer
-you don't have to perform WesternBlot, you can also to a Coomassie staining
- for Coomassie staining about 0,5 µg are ok, for WesternBlot take less (0,1 µg)
Degradation means, you will have two bands for the antibody: around 55 kDa and 25 kDa
Hi Gaurav
If ur mainly interested in checking the antibody degradation... then go head WB...
But if u want to check either ur antibody is functional. Then the easy way s DOT blot assay.
If the ab s not working in dot blot . it wont working in any kind of antigen antibody reactions.
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