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check of amount of proteins on filter after rouge ponceau|

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#1 jasmina



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Posted 09 June 2010 - 02:16 AM

Hi all,
I ran an SDS-PAGE gel of 2 different samples, 50ug each. Then, make transfet of proteins on nitrocel. membrane.
when, i stain by rouge ponceau, i see that the amount of proteins present in the 2 wells is very different. one line with many proteins, and the other with very very few, if even empty, ap art from one band in the middle of the line!!!!
i have quantified these 2 samples with bradford separately, not the same day!
does is mean that there is a less protein in one sample than the other, or the quantification of one of them that is incorrect!
thanks for your reply

#2 bob1


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Posted 13 June 2010 - 04:54 PM

There are two scenarios here:

1) that your quantitation is fine. In this case the protein bands you see in the lane with less bands should be very obvious compared to the lane with many bands. If this is the case, there is not much you can do, you can still work with these samples, it may be that this is exactly what you should see (e.g. if the samples are from different fractions of a cellular fractionation experiment where you would expect to see many bands for the cytoplasm, and fewer for the nucleus).

2)Your quantitation is wrong. I would suspect this is the problem. Luckily it is the easiest to test - repeat the bradford assay on both samples at the same time.

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