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What causes this offset band on RIPA gel?


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#1 polaris

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Posted 20 September 2010 - 09:05 AM

This is an autoradiograph of a RIPA gel. The band at the arrow will sometimes show this odd stepped appearance, sometimes it's smeared and sometimes it's just not there. Also the standards in the far right lane are somewhat diffuse. Any experience with this condition?

This is SDS-PAGE with reduced samples (DTT)...

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#2 Curtis

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Posted 20 September 2010 - 10:37 AM

take a look up at the loading area, your sample has problem. centrifuge your sample again and use supernatant. it happens to me sometimes also.

#3 polaris

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Posted 20 September 2010 - 02:07 PM

Thanks for the reply.

I do centrifuge prior to loading. I suppose I could do it longer and harder. Would your explanation fit the observation that this only seems to happen with the two proteins I'm interested in at around 68kd?

#4 madrius1

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Posted 21 September 2010 - 06:53 AM

Couple Curtis's explanation with Murphy's law and you'll get your answer!

But more seriously, this looks more like an artifact than like a band, to me. Moreover, all of the major bands in the middle lane show a "bend" on the right side. Maybe your wells were not clean enough? Also, are the chemicals you use for casting your gel fresh?

#5 polaris

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Posted 21 September 2010 - 07:23 AM

In lane 1 there should be a band at 68 and in lane 2 there should be a band at 67; these should be the two darkest bands on the whole gel. Reagents are about as fresh as they always are when the assay works... I rinse the wells with running buffer and then aspirate to clean them out before loading samples.

I'm thinking about the samples themselves. After immunoprecipitation the samples are eluted off of protein-A sepharose beads (in loading buffer with DTT) by heating in boiled water for 5 min., then they get frozen overnight. The samples are thawed at room temperature the next morning while the stacking gel is being prepared (resolving gel is prepared the afternoon before). When the gel is ready, we vortex and centrifuge the samples and then load.

I'm thinking that it wouldn't hurt to reheat, before spinning down and loading. Since I only see this problem with these two higher wt. proteins, could it be that they are more prone to forming some kind of precipitate or secondary structure at low temperatures?

Edited by polaris, 21 September 2010 - 07:26 AM.





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