Western Quantification (No BCA???) - (Mar/26/2013 )
Love perusing the forums, but now I have a need for some input from the peanut gallery.
I'm pretty concerned about a technique being used (er... skipped) in a lab that I've rotated in. For one reason or another, the technician(s) run western blots without doing BCA or Bradford. They sometimes* empirically determine the linear range for IR or ECL-based detection with serial dilutions to avoid overexposure, but their loading is definitely not equal across lanes.
The justification is that they always run samples at least in triplicate, and subsequently normalize each band to its loading control (on the same membrane) using commercial and/or free image quantification software (Odyssey and ImageJ). Now, I buy this to a certain extent. Provided that both the loading control (actin usually) and target protein bands are in the linear range of detection/intensity, the ratio between the bands can be compared statistically across many samples.
However, it runs contrary to everything I've learned to skip the BCA or Bradford - and considering how easy BCA is, it seems really odd to skip it.
Has anybody else ever seen this? I get the sense that the lab is somewhat recalcitrant to changing protocols... I think I might save some lysate and run BCA incognito if I am told off for wanting to do BCA
Thanks for your advice
it is not the best practice to not know how much they are loading (you don't want to overload the lane, it could affect the adjacent lanes) but, if they are looking only at a ratio of two or more proteins to each other then it may be acceptible.
I agree with mdfenko. It would be easier to do a bradford and determine your loading concentrations, but they may think it unnecessary if using IR or ECL.
These methods are more quantitative and if they do three titrations of there protein lysate, they can get a better overall comparison of the amount of there protein of interest in the sample. Why don't you just ask them? I hope they would be kind enough to give you an explanation.