Human cells were analyzed via MTT (abs. 570nm) assay before being lysed in 10%SDS and frozen. Will I be able to determine protein concentration in order to perform a western blot? Will the SDS affect lowry or bradford method of protein concentration determination? If not, how would I determine protein concentration between samples?
Do you need to know that (1) equivalent amounts of protein are loaded in each lane or (2) the absolute amount of protein that is loaded in each lane?
(1) If you normalized the samples based on their MTT at 570nm before freezing them down you can load identical volumes in each lane and assume the amount of protein is the same in each lane (if the cell samples are not very different). If you froze down equivalent sample volumes (without regard to their MTT) you can still load equivalent protein by scaling the volume added to each well based on their MTT. Internal controls like b-actin are often used to confirm equivalent loading of each lane in a western.
(2) Its much rarer to require the absolute amount of protein in each lane, but using a protein assay like the BioRad DC, which can handle high SDS, should allow you to get these numbers if needed.
SDS is not compatible with the Bradford assay, but can be used with the Lowry and BCA assays.
http://www.beyotime.com/Compatibility%20Chart%20For%20BCA%20Kit.pdf
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_1069.pdf
precise and simple method is BCA (Pierce kit ), it is compatible with samples that contain up to 5% surfactants (detergents).
I have been using BCA for my samples for years. All samples are solubilised with detergents. Its very simple and band look similar when I later probe with loading controls
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