4 replies to this topic

[ly07]

Hi, I have problem with my tris/tricine sds page gel recently. I have been using it for a couple of years.  I didn't run gels for a while. When I started to run 2 weeks ago, I found got this problem.  The dye front of the samples is diffused and uneven (not forming a straight line) and the dye smeared very badly.  The protein ladder runs nicely, focused well. The Dye smear interfered the low molecular weight peptides.
The gel is a standard 15% tris-tricine sds page.  I run it in the cold room.
It is very similar to the problem described here  "Trouble-shooting Tris-Tricine SDS-PAGE  http://www.protocol-...html"  I'll really appreciate your advice.

Edited by ly07, 08 March 2011 - 01:22 PM.

[mdfenko]

besides what i wrote in the thread you linked, old, somewhat decomposed, sds can also cause what you are seeing.

try using fresh buffers with a new batch of sds.

[ProteaKyle]

I have come up with a few other things you can check.  It might be that your Bromophenol Blue concentration is too high.  The most common concentration should be between 0.005% - 0.01%. Another thing to check is the sample buffer.  The shelf life of the sample buffer is about 30 days at 4°C, 6 months at -20°C, and 1 year at -80°C.  I saw that someone said that they had similar problems when their SDS precipitated out, I have never had this problem but I did read that SDS can precipitate out at low temperatures.  Since you are running your gels in the cold room then you can try replacing the SDS with LiDS.

I hope this helps you solve your problems.

Kyle
Laboratory Technician at Protea Biosciences
www.proteabio.com

[ly07]

Thanks for the responses.  I will check out these things!

[ly07]

I replaced SDS in sample buffer and running buffer.  The smear disappeared.  
Thanks guys!

Edited by ly07, 11 March 2011 - 07:28 PM.