SDS-PAGE: Strange Running - (May/20/2010 )
I've been getting this strange effect recently that I can't seem to figure out.
Basically I'm running a 15% Acrylamide Gel in 1x Tris-Glycine Buffer at 160V for 1 Hour. I've been using this protocol for over a year without too many problems until recently, where the lower half of my gel seems to run extremley poorly (pic attatched). I've re-made gels, used pre-cast gels and re-made my running buffer and still get the same effect. Unfortunately my protein of interest is in the size range that is obscured by the poor running!
Any suggestions???
am i correct in assuming that you ran sds-page?
if so, when you remade your buffer did you use the same lot of sds?
if so, try remaking your buffers with a fresh lot of sds.
First of all, I think that 160 Volts is too much. Do you have any ice in the tank when you run the gel? The gel gets heated up and causes trouble.
I would run it slower, start with 120Volts and go up to 150 Volts when the front reaches the resolving gel. The slower you run it the better.
The only time when I personaly had this trouble and my gel looked like yours was when I added too much SDS in the loading buffer.
Is it maybe this the case?
Cheers for the replies.
Yeah I did use the same SDS, I will definitely give it a try remaking the gels and buffers with new SDS. I don't think it's my loading buffer as I made a load of it about 6 months ago and aliquotted it. It's being working fine before with this batch of loading buffer so I think it's okay.
Will let you know how I get on!
how do you store the aliquots?
if you store at 4C or below then you must ensure that sds is back in solution prior to use (sds crystallizes at low temperatures).
you can try a different aliquot of your loading solution to confirm that it is still okay.
I had the same problem with my lower half of the gel and remade my 1.5M Tris-HCl (pH 8.8) and my 10% SDS and my problems went away..So definitely remake those stocks and make sure your pH is where it should be in your 1.5M Tris-HCl solution.
Good luck
TomH on May 20 2010, 07:57 PM said:
Basically I'm running a 15% Acrylamide Gel in 1x Tris-Glycine Buffer at 160V for 1 Hour. I've been using this protocol for over a year without too many problems until recently, where the lower half of my gel seems to run extremley poorly (pic attatched). I've re-made gels, used pre-cast gels and re-made my running buffer and still get the same effect. Unfortunately my protein of interest is in the size range that is obscured by the poor running!
Any suggestions???
i seen ur gel pic
i keep having the same prob on and off. i run my gels at a constant current of 300mA along with an ice pack in the tank and on a magnetic stirrer. still the prob persists. its very random. does anyone have an explanation for this and a solution would be most welcome.
i have even used fresh buffers and still i am never sure when the and why the problem returns
Is it possible that your protein samples have not the same pH? Do you treat them the same when you isolate protein? Do you wash them all the same with PBS? Is there ANYTHING that you do differently?
Turned out the problem was the SDS as suggested! Many thanks to all and my gels are now running beautifully!
