We have been working on metaproteomics of bacterial community. On 2D electrophoresis we are unable to resolve protein spots. Our protein spots remains clumped on gel as streaks. 50 micro gram of proteins were dissolved in Destreak rehydration buffer (GE Health Care) on 7 cm IPG strips (pH 3-10). For second dimension we used 12.5% SDS-PAGE. On silver staining , we could not obtain seperated spots. All the spots seems to be clumped together. What could be the reason for that?
Is this the problem with our sample preparation or is it due to our strip length
Please suggest.
Gel picture is attached herewith. Thanking you
Regards
Divya jose
Hi Divya,
There are multiple reasons for that:
1. Technical - sample prep, gel quality, stain quality, very high salt concentration.
2. biological - excess of glycoprotein (which leads to streaks), highly diverse protein population that leads to low quantity of individual proteins and so on.
Did you run a test sample (like E. coli extract) to make sure your methods are good?
Cheers,
David.
Agree with David on all of the above. Bacteria can be tough to work with so you will need to optimize your sample prep and protein extraction.
Can you get a decent 1D gel in SDS sample buffer?
Jens
Hi Jose,
Did you precipitate your proteins before re-suspending it in the 1st dimension buffer?
Cheers
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