Laemmli buffer - yellow color (Feb/17/2010 )
I prepared by reduced Laemmli buffer some time ago and at the beggining it was blue, but then with timw it started to lose the color and now is almost yellow/orange but when addign to some protein it becomes again blue??? why it is happening?? soem pH changes ?? can I use it still??
Thx
-porfirion-
porfirion on Feb 17 2010, 11:26 AM said:
I prepared by reduced Laemmli buffer some time ago and at the beggining it was blue, but then with timw it started to lose the color and now is almost yellow/orange but when addign to some protein it becomes again blue??? why it is happening?? soem pH changes ?? can I use it still??
Thx
Thx
pH changes possibly. I know when I resuspend TCA precipitated proteins in sample buffer, it will get green, and a few drops of 5 M NaOH fixes it. Not sure if that should be done in your case. There might be degradation of a component that occurred as well as a pH change. You could try to add some NaOH to it to see of the pH changes, and try using it with some unimportant samples to see if it still does its job.
If it were me, however, I'd just make up some new buffer.
-fishdoc-
Seconding pH change, acidification of bromophenol blue turns it yellow.
-bob1-
fishdoc on Feb 17 2010, 12:31 PM said:
pH changes possibly. I know when I resuspend TCA precipitated proteins in sample buffer, it will get green, and a few drops of 5 M NaOH fixes it. Not sure if that should be done in your case. There might be degradation of a component that occurred as well as a pH change. You could try to add some NaOH to it to see of the pH changes, and try using it with some unimportant samples to see if it still does its job.
If it were me, however, I'd just make up some new buffer.
If it were me, however, I'd just make up some new buffer.
you use 5M naoh? that's a little harsh. we use 1M tris pH 9 to neutralize the sample and return the color to blue.
-mdfenko-
mdfenko on Feb 18 2010, 09:34 AM said:
fishdoc on Feb 17 2010, 12:31 PM said:
pH changes possibly. I know when I resuspend TCA precipitated proteins in sample buffer, it will get green, and a few drops of 5 M NaOH fixes it. Not sure if that should be done in your case. There might be degradation of a component that occurred as well as a pH change. You could try to add some NaOH to it to see of the pH changes, and try using it with some unimportant samples to see if it still does its job.
If it were me, however, I'd just make up some new buffer.
If it were me, however, I'd just make up some new buffer.
you use 5M naoh? that's a little harsh. we use 1M tris pH 9 to neutralize the sample and return the color to blue.
No, you're right. That is harsh. I keep a volume of 5 M on my bench, so that's why I thought 5 M at first. I diluted it 5x or 10x before using it. My bad. And rather than it being "a few drops", it was a few microliters. I think 5 or 6 ul in a 100 ul sample returned the color.
-fishdoc-