Hello every one,
I'm facing problem in detection of particular protein of interest in TMA of prostate cancer. After adding the DAB solution the brown coloration is not coming. So I'm not getting any signals of my protein of interest. We used Streptavidin conjugated secondary antibody for Signal amplification. So any one can suggest me where is the problem.
Dear Prakash,
I just want to make sure that I am understanding you correctly. You added a primary antibody to recognize your protein of interest, and you then added a secondary antibody (that recognizes the primary antibody) that has a streptavidin tag conjugated to it? Is anything else attached to the streptavidin?
For amplification I prefer to use a biotinylated secondary antibody, and then add streptavidin conjugated to either HRP (for DAB) or a fluorescent tag. If this is actually the technique that you are using, and your positive control slides show no DAB signal, then check your hydrogen peroxide. It can go bad very easily, particularly the 30% stock.
Cheers!
Jill
I agree with Jill, other than using the wrong antibody (I'm still ashamed over that) the hydrogen peroxide is the easiest way to get no results. I would do a iodine-thiosulfate titration, you can use permanganate, but the filtering of MnO2 is pretty time consuming and iodometry is simpler IMHO.
Dear Jill,
Thank you for your valuable suggestion. We are also using biotinylated secondary antibody, followed by addition of streptavidin conjugated HRP. I will look into the hydrogen peroxide. Is there any other possibility of getting no result?
Thanks,
Prakash
Dear Prakash,
HRP is a pretty hardy enzyme, so I would check the DAB and Hydrogen Peroxide first. Purchasing new H2O2 is a very cheap fix. On occasion, I have gone to the drug store and purchased the 3% that is available over the counter to use when my 30% was questionable and I needed to perform the reaction that day.
Do you have access to any of the other substrates that can be used with HRP? You could use one those to rule out whether your DAB has gone bad, although sometimes they are not as sensitive.
If you have new substrate that you have tested (you can just add it to a small aliquot of your streptavidin and check for the color change), and you still have no signal on your positive control tissues, then it is time to figure out whether your secondary antibody is not recognizing it's target. To check that, you would need a reliable primary antibody and control tissue. I am a neurobiologist, and in our lab we have a very reliable anti-neurofilament antibody that we use for this purpose.
Good Luck!
Jill
Dear Jill,
Thank you. we don't have access to any other substrate but I will go for a different peroxide stock.
Thanks,
Prakash
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