SDS-PAGE HELP --MISSING DESTAINCONTROL - (Sep/28/2001 )

What is a destain control ? What is its purpose ? If your buffer has KCl in it and this buffer is sued to suspend BSA in it prior to electrophoresis in SDS, BSA will not show up.

Hijda Anil Tyagi

Hi, I am having a bizarre problem with an SDS-PAGE. I'm running a gradient gel (10% - 20% Acrylamide) using 1M DTT as the reducing agent. My problem is with the destain control (0.01 mg/mL BSA) not appearing on the gel. I have been running this gel for well over a year without a problem, and all of a sudden, the destain stops showing up. All equipment has been checked, all reagents have been systematically changed to new lots, and everthing has been made correctly. All proteins and markers look fine on the gel. Except for the destain, the gel looks beautiful. The problem occurs with different analysts in two different labs. We have used different lots of BSA and checked with vendor to verify any procedural changes on their end, there were none. I have run a gel under same conditions with 5 different destains and markers. All looked fine. Another analyst ran the same type gel on the same day with the same reagents and destain, and the destain did not show up. Has anyone ever seen anything similar? Any help would be greatly appreciated. Melissa

Hi. The destain control is just 0.01 mg/mL BSA in water that we put on the gel to insure that we are not overdestaining and possibly losing any small bands that might be present in our samples. We take 12.5 uL of the BSA, add our reducing buffer and run it along with the samples. I do not believe that the buffer contains any KCl, but I will check. We have been using it for years and all of a sudden...poof...it's not showing up anymore. However, our molecular weight marker contains BSA (not sure of the concentration). It is made with the same buffer as our destain control and it shows up fine. It's a real mystery. Thank you for you time. Melissa

- Maybe you did over-destain the gel and/or did not stain long enough