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study protein expression in bacteria

protein expression bacteria

Best Answer bob1, 01 February 2016 - 11:52 AM

You can store pelleted (and washed) cells at -20 or -80 (think stability of protein) and thaw for lysis. I would recommend  pellet the cellst, wash, aliquot, then pellet again so that you have individual samples. However, note that if you need to try different growth conditions, this is not the best approach as for a bulk culture the growth will be uniform within that culture.

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#1 Meg P. Anula

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Posted 01 February 2016 - 01:20 AM

Hi.

 

This is the first time I'd be doing protein expression study (western blot) of a recombinant bacteria.

 

Normally the steps would start from streaking the recombinant bacteria stock, pick one colony and grow in 1ml broth overnight. Then spinned down the cells, washed with PBS and resuspended in 500ul PBS/PMSF before sonication. Cells lysate is then mixed with 5x sample buffer and boil for 10 min.

 

The samples are then spinned down (13000xg for 3 mins) and lastly the supernatant is collected to be resolved by 12% SDS page.

 

Since I am still at the beginner stage for SDS and WB, at lot of optimization works need to be done.

 

So I am thinking do I need to repeat from streaking and growing bacteria broth culture o/n every time when I wanna repeat the whole steps. Or can I store a large amount of broth culture in -20c and use a small amount each time to test on the protein expression.

 

TQ.


Edited by Meg P. Anula, 01 February 2016 - 01:21 AM.


#2 bob1

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Posted 01 February 2016 - 11:52 AM   Best Answer

You can store pelleted (and washed) cells at -20 or -80 (think stability of protein) and thaw for lysis. I would recommend  pellet the cellst, wash, aliquot, then pellet again so that you have individual samples. However, note that if you need to try different growth conditions, this is not the best approach as for a bulk culture the growth will be uniform within that culture.







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