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

Western Blotting Protein SDS PAGE Electophoresis Coomassie blue

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#1 GraduateStudent2015

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Posted 03 March 2015 - 03:42 AM

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 ! 



#2 mdfenko

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Posted 03 March 2015 - 04:46 AM

are you sure you're not getting false protein concentrations due to buffer components?

 

you can try silver staining the gel (although coomassie should have shown the major protein bands if you loaded 30ug).


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#3 GraduateStudent2015

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Posted 03 March 2015 - 05:05 AM

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 ?



#4 mdfenko

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Posted 04 March 2015 - 05:28 AM

in what do you suspend the protein pellet? if rlt then what is in rlt?

 

did you run a buffer blank with the bca?

 

you can try washing the pellets after extraction and compare the results with those from ripa samples if you wish.


talent does what it can
genius does what it must
i do what i get paid to do





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