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SDS-PAGE run problems


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8 replies to this topic

#1 cuca154

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Posted 18 January 2005 - 07:56 AM

I've been running some protein samples in SDS Page and getting run problems.
Although everything looks fine (voltage 200V etc) the samples donīt seem to get out of the wells. Indeed after some time the dye begins to blur and disapear. After almost an hour of run I donīt even see the front line.
Any ideas?
Thanks a lot.

#2 thefallguy

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Posted 18 January 2005 - 08:10 AM

The resistance in your system could be very high. This could be due to a damaged electrode or a method of sealing you may have used for the bottom of the vertical gel. Have you tried changing the apparatus?

#3 cuca154

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Posted 18 January 2005 - 08:13 AM

Its a brand new BioRad Protean 3...

#4 thefallguy

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Posted 18 January 2005 - 08:19 AM

Cuca

Brand new apparatuses often have conductivity issues. Make sure there is current flowing through the system. A Tris-glycine buffer filled system should show at least 25 mA at 100 V- if not, something is wrong.

#5 cuca154

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Posted 18 January 2005 - 08:55 AM

Thefallguy,

It has 125 mA.
Is it possible that buffer pH could induce this? I've just measure them again and they are a bite lower than they should...
Thanks a lot.

#6 thefallguy

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Posted 19 January 2005 - 06:36 AM

Cuca

The current is too high. This suggests that either gel conductivity is high because of ions in acrylamide (improbable - but happens once in a while) or there is another electric contact being made between buffer reservoirs separate from the gel sandwich (highly possible). Your samples donot move because all the current is not passing through the gel - it is being diverted somehow. Try this- remove the gel sandwich- connect apparatus to power. If the current is still at 125 mA at the set voltage - then the gel is not seeing the current at all.

Good luck

#7 thefallguy

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Posted 19 January 2005 - 06:39 AM

Cuca

If the buffer is bad- then the gel will either run slow or very fast - but not diffuse like you describe. If I were a gambling person, my money would be on the apparatus, not on the buffer or your samples.

#8 cuca154

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Posted 20 January 2005 - 07:39 AM

Thefallguy,

I did what you mentioned on your last post and as you predicted it do seems that the current is not passing thru the gel!
I'll just have to check the apparatus for the problem... :D
Thanks a lot, you were very helpfull!

#9 thefallguy

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Posted 20 January 2005 - 09:18 AM

Cuca

You are very welcome. All we have is each other in this often times very difficult profession.




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