question about denaturing - (Oct/24/2010 )
I am a beginner of western blot with some general questions.
1) since the protein will be degraded somehow. could I just boil the sample after lysate (in RIPA buffer)? Will this protect the protein from degrading? there are some sample preparation protocols online used sample loading buffer for lysate and then boil the sample right after. not so sure if we can boil the sample just in RIPA BUFFER.
2) if i want to use sample loading buffer for protein preparation, for example, 2x leammli buffer, how can I dilute it back to 1x for loading? or it really does not matter.
any help will be appreciated.
1) ripa and sample buffers are not the same. boiling in sample buffer assists the denaturing process of sds and reducing agent (if present). you can heat at lower temperatures (ie 60-70C) for 10-20 minutes to avoid aggregation of the sample.
2) adjust the final volume of the sample to 2 x the volume 2x sample buffer used. with 2x buffer it may not make a significant difference but you can ensure that all samples are treated equally if you do the final dilution.