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GraduateStudent2015's Content

There have been 2 items by GraduateStudent2015 (Search limited from 03-April 19)


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#173640 Problem with Western Blotting - BCA assay shows protein but no protein bands whe

Posted by GraduateStudent2015 on 03 March 2015 - 05:05 AM in SDS-PAGE and Western Blotting

Its essentially an acetone precipitation with EtOH wash step. I assumed that the protein pellet would be free of most salts. Also, my samples had varying levels of protein so i assumed that the RLT buffers were not interacting and giving me false readings. Maybe I should try washing the pellets throughly after extraction and comparing results from RIPA lysed samples ?




#173638 Problem with Western Blotting - BCA assay shows protein but no protein bands whe

Posted by GraduateStudent2015 on 03 March 2015 - 03:42 AM in SDS-PAGE and Western Blotting

I recently harvested protein using the appendix protocol from Qiagen mini RNA kit extraction kit. I have mammalian tumor cells cell lysate from my samples (Very low number of cells >million cells) therefore need to use a single method to extract RNA and protein from the same sample. I ran a BCA protein quantification assay and had good protein concentrations. However, when I checked my SDS PAGE gel after running it at 180v (4-20% gradient gel) for less than an hour (Protein ladder ran till the bottom of the gel) using coomassie blue staining I saw NO bands on any sample lane. I stained using microwave hot Coomassie for an hour. I had according to my calulations 30µg protein per lane. Shouldnt heavily expressing proteins like GAPDH/beta actin/integrins show up quickly on coomassie ? Why am I getting excellent protein quantity using BCA but cannot see anything on my gel. THere is no smear or faint bands so I cant confirm and say its degraded protein. Please help ! 





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