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western blot and actin issue - (Jan/10/2013 )


I would greatly appreciate any help. I recently infected some mammary epithelial cells and harvest the protein. i use the quibit quantification to determine the protein concentration of all my samples. i ran a western blot and probed for b-actin. the b-actin were not equal. i have had trouble in the past with the quibit and nanodrop. i was wondering if i can just chose a volume of protein (not equal conc based on quibit quantification), run a western blot, and then use imaje j to adjust the volume? So if i get all the density form image just can't i just standardize them to one of value and change the rest or is there not a linear relationship between protein concentration and band intensity? thanks.


by saying 'the bands are not equal' you mean the thickness of the bands is not the same? we usually use microplate for Bradford method to assess protein concentration and read the values under a microplate reader. I'm not friendly with using Nanodrop for protein concentration mainly because it would be difficult to run standards one by one and check several samples, although the newer Nanodrops are multichanneled. We normalized our samples to have 15 to 50 ug of total protein for each well of BioRad's PROTEAN mini set.


A few comments and questions to try and help you.
First, when you say they are not equal... how different are they? Is it small or clearly significant.
Second, do you ponceau your membranes before antibody incubation? If so, how did your cell lysates looked (I assume you are running lysates). If you have seen a clearly significant difference in you actin levels, and this is due to different protein load, you might be able to see (not quantify) this by ponceau.
Finally, are you sure that infecting the cells doesn't affect actin expression? I know Actin is widely use as a control, but is not necessarily the best one, or valid for every experimental setting.

-almost a doctor-