I runned twice the western SDS Page with my protein samples in 4x lamelli buffer (diluted to 1x with 1X TBS in protein sample lysate) but despite getting blue front of bromophenol blue. I found yellow color of it after few minutes as it crossed resolving gel with bad smile. I checked my buffers pH of running buffer -PH-8.3, resolving gel ph 8.8, stacking gel ph 6.8. but while searching I found that yellow color is due to acidic ph in that region even that region ph of resolving gel Tris base is 8.8.
Please help me troubleshooting it...
You have identified right, Bromophenol blue serves as a pH indicator. It turns green at acidic pH. As you already checked your buffers, you can make fresh running buffer and use that for making gels. Make sure your TBS is fine. Also, the dye that you are using right now, change that too. If its not the buffers, may be its the dye. Does it remain blue in stacking and only turn green when your proteins enter the resolving gel? Then may be you should prepare Tris pH 8.8 buffer fresh and see if that happens again.
Hope it helps!
Hey
To avoid smiley effects I fill up ALL pockets with at least some loading dye oder some BSA, if you do not have sample for all pockets. Then all lanes are filled with proteins and this helps with the "smiley".
Cheers
I've seen this type of "distortion" due to heat build up in the gel, and also due to high salt concentration from my samples.
As you, and others, correctly assert, the colour change is pH dependant. The yellow colour suggests a pH
Their might be a problem in your sample.
Have you did desalting?
Check the pH of your sample.
Run the gel in low temperature (2 to 8 deg).
Thanx shiyam! My samples are fine as checked from staining with commasive brilliant blue dye soon..beautiful bands appeared on gel..! Means samples donot have any problem
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