I have been working with whole yeast extract to search for proteome using two-dimensional electrophoresis. Here, I have attached three silver stained gel images acquired with the similar procedure. However, two gels are showing streaks (both horizontal and vertical), can anyone come up with the exact reasons?
I would wonder more on why second gel is very nice compared with the first one, they must be some differenceses in the sample or protocol
Streaks in second gel not important and would disappear if you no not overstain the gel
Hi,
Horizontal streaking (parallel to the IEF strip) is known to be caused by incomplete focusing in the first dimension. The time required to complete the first dimension can differ between samples, so it is advisable to monitor that properly (for example check when the current stabilizes).
Hope this helps!
Thank you all for your suggestions.
@Eef Dirksen: I have monitored the current, which is stabilised @50uA, however, it gets decreased to 30uA after 4000V reach but is still stable. Whereas voltage initially does not reach to 500V and 4000V in time but after that, it goes okay.
Hi...
In the second gel you have tears more than spots...
Increase SDS concentration in the upper chamber will solve this. You can use thw running buffer 2X or even 3X in the upper chamber.
Orhers gels seem to have a lot of contaminats...use the maximum quality reagents.
Good luck
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