Dear All,
I got low recovery of high MW protein from my 2D gel. Please check the attached picture.
I used 13 cm IPG strip and loaded 300 ug Hela protein. 7M Urea and 2M thiourea were used in the lysis buffer and rehydration buffer. Gel was equilibrated for 30 min with DTT buffer and 30 min with iodoacetamide. Each buffer has 2% SDS.
IPG strip is from GE Immobilian Drystrip pH 3-11 NL 13cm.
Slab gel is Biorad Criterion XT precast gel 4-12% Bis-Tris for 2nd dimension.
Can anyone give me some suggestions?
Thank you very much!
Hi Feng Jin,
I think you have, a problem called SDS depletion, It s caused by a too low SDS concentration. Therefore not all proteins (especially the high MW proteins) are saturated with SDS, and this makes the spot-rocktes you are seen in your gels.
This can happens because in the upper buffer chamber, there is not enough SDS for all the run. Try to use 2X or 3X SDS in the upper buffer chamber, and your results will look better.
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