You cannot determine protein contents. If you had already protein contents, it may be a distorted measurement due to the presence of debris, aggregates and varying membrane vesicles ruptured from the cellular and organellar membranes. However, you could overcome by adding 2xSDS sample buffer and rock the tube for 1h at 4C to dissolve proteins as much as possible. Spin it down at 12,000 rpm and take the upper part to avoid mix the precipitates. Make aliquots in tubes if you have a lot. Store at 4C. Prepare new stock of the same cells with proper spinning down this time. Make sample for SDS-PAGE as above (you have measured protein contents at this time). Run the gel with your favorite protein contents loading on 1st lane and 5ul, 7.5ul, 10ul on lanes 2, 3 and 4, respectively. Coomasie B blue staining the gel. You could determine how much of your first sample should be loaded for optimum protein contents by comparing with the first lane of known protein contents.

5 or 7.5 or 10 or 12.5ul for your Western blotting next time?

Best wishes

Well, it depends on the method, but in principle yes, for two/three reasons

1) the dye may bind to rests of organelles etc. that would be otherwise removed

2) especially Coomassie is affected by micelles, thus present lipids and other substances may affect the measurement

3) turbidity of the sample will increase what you read as absorbance

dear Daniel,

I think there is no problem if you collect your protein from the store and do centrifuge after that collect the supernatant. otherwise you have to make new lysate.

Hi,

If you have more lysate, spin it down and then run the gel again.

Thanks so much