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"Wavy" bands in SDS-PAGE gel

SDS-PAGE uneven bands MHC myosin heavy chain electrophoresis

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#1 bvelten

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Posted 19 July 2013 - 05:32 AM

Hi,

I've been running SDS-PAGE gels this summer for the first time trying to separate myosin heavy chains present in skeletal muscle. I've noticed that sometimes I get uneven and wavy bands. Sometimes this is actual curvy-ness in the band, sometimes it's just the band tilting in the lane. I can get gels where I have no waviness at all, but I haven't figured out what I'm doing differently. Is the waviness just due to tearing the wells? Or possible bubbles at the bottom of the separating/resolving gel?

For my gels, I run two gels at the same time on an Hoefer SE600 system. Because I'm separating such heavy proteins, I run the gel at around 7C for about 45 hrs. My voltage in the stacking gel is 70V and then I increase it to 200V in the separating/resolving gel.

Here are two gels I ran yesterday, both illustrating my issue:
7-16-13 gel1 1.jpg
7-16-13 gel2 1.jpg

Thanks for all help!

#2 Inmost sun

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Posted 19 July 2013 - 09:39 AM

if it is a crude extract it may contain salt which disturb performance of SDS-PAGE

Edited by Inmost sun, 19 July 2013 - 09:39 AM.


#3 bvelten

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Posted 19 July 2013 - 09:45 AM

But it doesn't always happen with the same samples.

For example, in the first gel image, lanes 3, 7, and 13 are all exactly the same thing, but only lane 7 has a wave.

#4 casandra

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Posted 19 July 2013 - 10:05 AM

We already have a few discussions about this in the forum…here’s one: http://www.protocol-...osts/22295.html

You can also check out this SDS-PAGE “Hall of Shameful Gels” http://www.ruf.rice....e/sdsgoofs.html
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#5 bvelten

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Posted 19 July 2013 - 10:16 AM

Thanks for the tip. I had seen the hall of shameful gels before, but I'm fairly certain my stacking/separating interface is clean and straight. I also rinse out my wells with running buffer a few times before loading.

I'm going to try letting the gels polymerize for a bit longer, as well as ensuring wells are straight and I don't remove the comb to quickly or roughly. We'll see if that helps.

Thanks for the tips.

#6 mdfenko

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Posted 19 July 2013 - 11:13 AM

in addition to the advice in the linked post, if you are going to run the gel at 7C then you will have to use lithium dodecyl sulfate (lds) instead of sds. sds crystallizes at lower temperatures and can cause all sorts of mischief.

we used to separate myosin heavy chains (from brain) by 3.5-5% gradient gels with an 8-0M urea gradient overnight runs at 5mA (i think, it was a very long time ago).
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