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Trouble-shooting Tris-Tricine SDS-PAGE


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#1 ly07

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Posted 08 March 2011 - 11:51 AM

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-...sts/41821.html" I'll really appreciate your advice.

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


#2 mdfenko

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Posted 09 March 2011 - 01:28 PM

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.
talent does what it can
genius does what it must
i do what i get paid to do

#3 ProteaKyle

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Posted 10 March 2011 - 10:59 AM

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 4C, 6 months at -20C, and 1 year at -80C. 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

#4 ly07

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Posted 10 March 2011 - 07:12 PM

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

#5 ly07

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Posted 11 March 2011 - 07:27 PM

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

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





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