I have been struggling in trying to localize the protein of interest i detect by 2D immunoblotting on duplicated silver staining.
i tried to duplicate the 2 D gel and use one for 2D Immunoblotting and the other one for silver staining. and every time i fail to localize the spot due to many complexities such as marker protein load differing in levels between 2 gels and non identical reproduced pattern ....
if there is anyway to visualize the proteins before transferring them to NC membrane it will be great so that i can match this immunolabled protein with the protein before labeling and matching through the total protein population to reconize the pattern. i tried ponceau red protein staining but it's not sensitive enough.
any suggestions?
Honestly dind't understand the question...
Do you know what protein you're looking for (its molecular weight and pI) or that is precisely what you're trying to figure out?
Have you successfully spotted it in some gel/immunoblot at least once?
Develop some more: what protein, what immunolabel, what sample, ...?
Ok. sorry i didn't explain enough. the protein is unknown. i am using total protein extract from bacteria to detect argpyrimidine residues using argpyrimidine specific Ab. i found protein. and i want to isolate it to send it for fingerprinting. but i can not isolate it from membrane as it must be isolated from gel. but the problem is that i can not localize same protein on silver stained gel of the same sample due to the error of determining same spot observed on 2D WB.
Sometimes you can't find your protein of interest in the gels with silver or Coomassie blue staining as their sensitivity is quite low when compared to the WB antibody signal, thus the spot you identify with your antibody can be very faint in the gel.
You could try running new gels with a higher protein loading, 2 - 3 x more, that might help to increase the protein concentration for each spot, and maybe it will give you a better signal after the silver staining.
Hi,
I think I get your problem and this is indeed very tricky.
I agree to the answer above from R Trevisan that the 2 maps you get from your proteome must not look the same due to the sensitvity, so increase the protein load.
That might be totally silly but it just crossed my mind: load as much protein as you can and start the transfer of the protein onto the membrane but do not make it complete so that you have proteins both on the membrane and still in the gel to match after staining/probing.
good luck!
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