Problems on IEF for 2DE - poor resolution near pH 10 (Apr/06/2004 )
I'm using the 7 cm IPG strip and IPGphor system for the first dimension of 2DE. Recently, I have purchased the 7 cm IPG strip holder for active rehydration prior to IEF. But I got (seems to be) a poorly focused 2D gel near the pH 10.
My protocol is, briefly, active rehydration in 7 cm IPGstrip (pH 3-10L) for 16 hrs and then IEF for 16000 vhr. Damp electrode pads were removed after voltage reached 1000V.
However, there are a series of spot appear vertically along the gel near pH 9 to 9.3...can't reach the edge of gel (edge of IPG strip)
Also, there are several horizontal streaks in the region near pH 10, besides the "column" of spots. I suspect that these streaks are proteins that could not be focused...which is outside the electrode.
So I would like to know why and how to solve the problem. I found that the gel length of IPG strip (7 cm) is longer than the distance between the electrode in the holder (~6.1cm). then, can the sample in the gel migrate to its pI (if it is very close to pH 3 and 10). And can the sample in soaked in the gel outside the electrode be focused to its pI??
Finally, what could be done except using the cup loading strip holder for IEF.
THanks for any opinion
IPGPhor capabilities break down near pH 10 probably because the strip composition assumes that target proteins might be more prevalent between pH 5.5 and 8.8. Can you try the more traditional BioRad apparatus? It is more pliable when it comes to extreme ranges.