Protein expression (View forum version)



bhavana

Posted 03 September 2010 - 04:03 AM

Im expressing a 37kDa protein, when im seeing its expression in SDS PAGE ....im observing the expression at approx 30kDa. can anyboby suggest me a reason for this problem.

mole

Posted 03 September 2010 - 05:29 AM

Hello,

Check for truncation of your recombinant protein, As if you have N terminal tag u will get both truncated and untrancated protein. SO please check your sequencing results for a mutation which leads to Truncation or Try to grow cells at lower temperature and Induce with lower IPTG or other inducer.

With Regards,

Mole .

mdfenko

Posted 03 September 2010 - 08:20 PM

sds-page can give you a good estimate of your proteins molecular weight but is not absolute. various factors can influence mobility (eg amount of sds bound, phosphorylated sites, etc).

bhavana

Posted 11 September 2010 - 08:48 AM

His- tag is at C-terminal, there is no truncation in the sequence, im trying to express at 30deg even though im seeing band at 30kDa.

kingswill

Posted 14 September 2010 - 07:27 AM

Protein ladder gives you an estimate of the size of your protein but not absolute. What % is your SDS-PAGE? You may try higher % gel to give you a better resolution of proteins as that size.

Another possibility is phosphorylation which may cause an upshift in your gel. Bacteria contain machineries that may phosphorylate your protein from human or whatever species. You can dephosphrylate you sample and see if the band shift back down.

bhavana

Posted 21 September 2010 - 09:09 PM

im running 10% gel, expected band is at 37KDa

Prep!

Posted 21 September 2010 - 10:09 PM

are running a reducing gel? if yes then try running a non-reduced gel too!!! or vice versa!
why dont u try a western... may be the 30 kDa protein is a major contaminant if its not your product!!

bhavana

Posted 22 September 2010 - 07:49 PM

I have tried western also with anti-his antibody, im seeing a band at 30kDa

Prep!

Posted 22 September 2010 - 07:52 PM

what kind of sample are u using? reduced or non-reduced??
as pointed out earlier... SDS-PAGE is a best estimate of the molecular weight but not necessarily the accurate one!!

reason of editing: spell-check!!