GST + 6kD protein expression? Doesn't seem to be working! - (Jul/26/2006 )

Our GST fusion protein doesn't appear to be expressing our small 6kD protein. We know we have it inserted in the vector in the right ORF and the right direction, but when we express, the band on the PAGE gel is the exact same as regular GST. Is 6kD not enough extra weight for a band to be resolved? Or is there some way that our protein isn't being expressed at all, or cleaved after expression...or what?? Any ideas or solutions would be appreciated!!

Thanks!

Jess

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It could depend on the percentage acryl of your gel. Cannot you do a WB using an Ab raised against your protein? What if you try a 4% acryl gel?

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We don't have an Ab specific to our protein. We're actually toying with the idea of scrapping the GST and doing a fusion protein with His or FLAG.

We havent tried any other gels, but ours are 4-20% gradient gels.

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You can either use 12% SDS-PAGE or try 10% discontinue native gel with GST run next to it, you should be able to resolve 25 kd from ~31kd bands by SDS gel, or by charge/size difference by native gel.

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We were able to see a 5kDa peptide on GST using 12% gels, so the 4-20% should also be fine.
When you say you have confiemred the insert in the vector etc, was that done since you transfected your cells, as well as before? (Some bugs are nasty litle beasts and have been known to spit out inserts...don't ask me why, they just do sometimes!). If y ou'er unsure, try transforming again int oa different cell strain and see if you have any difference.

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Hello,

you should keep in mind that GST fusion proteins very often degrade, so that your protein is cleaved off from the GST.
Did you directly compare your expressed protein with GST on a gel?

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I forgot GST is so "small"! Then you should definitely be able to distinguish between 25 and 31! I don't have so much experience in this but do you think it would be possible to stain your gel with Comassie and try to see if you have a 6KDa product in addition to your GST? That colud tell you at least if the protein is cleaved

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hi,
on a 12% gel if u run proteins for little long time u can see clear difference between bands of gst alone and gst with ur protein of interest.
for me it looks like there is something wrong with construct hence bacteria does not express it, because immediately after induction ur running gel, hence one wud not expect quick degradation of GST. if it degrades also u shud see atleast a fraction of complex on gel.
try expressing as his tag fusion protein.

regards
payeli

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Thanks everyone for the info.

I think they checked the insert when they were looking for cells that had the insert in them, then they made the protein once and froze the rest. It was actually some else's project and we took over, so I guess it could be feasible that our insert was spit out, or it could be that there was fast degradation. There were a couple gels were it looked like we had very faint bands of our fusion protein, so we thought we'd let the cells incubate longer at lower temps, but still didnt get anything.

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I think you'll have to check the plasmid for yourself. dod a miniprep and PCR to see that your insert is still there.
While you have a supply of plasmid, try putting it into a different strain of bacteria. That way you might see if the cells you used last time are doing anything nasty to your construct.

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