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Sample does not sit in the well despite (glycerol checked)


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#1 pobrien

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Posted 27 March 2012 - 09:50 AM

I have recently moved to a new lab and I'm performing some SDS-PAGE and I am having issues with loading my samples into the well. As soon as the sample leaves the pipette, the sample escapes and floats to the top.

Initially I had thought that the glycerol concentration was not high enough but even after increasing the amount of glycerol in the samples, I am still having issues loading the samples.

The only other times I have had this problem it was due to DNA in the sample making it 'gloopy', which is not the case here as the samples were thoroughly sonicated during lysate preparation.

Does anyone have any ideas on what factor may be causing this? Can high levels of salt in the sample can cause this? I've asked around in the lab and no one can seem to help however they have observed this phenomenon before in the lab.

My protein samples are <1.5mg/ml btw.

Thanks!

#2 bob1

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Posted 27 March 2012 - 11:32 AM

Is there any chance you have some solvent contamination, such as ethanol or acetone, these will cause samples to float.

Another option is that you are accidentally using a concentrated buffer for running (e.g. 10x) instead of 1x, which would probably make your samples less dense than the buffer, so they would float.

#3 pobrien

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Posted 27 March 2012 - 12:28 PM

Thanks for your response!

I'm confident that there is no solvent contamination in the sample. The loading buffer that I use is a 10x solution with 20% glycerol content so for the samples I add 10 ul of 10x per 90 ul sample prior to vortexing/boiling. Initially I added additional 10xLB to try and make the sample load (by increasing the glycerol content) however that did not work.

I am going to test whether it is in fact the 10x LB thats the problem, rather than the sample, by making up 1xLB in water and seeing whether it loads properly. I'll also make up fresh LB using a different method as the current lab that I'm in make up 10x where as my previous lab made up 5x LB. Another difference is that the current lab have DTT already present in the 10xLB rather than adding it fresh however I doubt this contributes to inefficient loading of sample.

#4 mdfenko

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Posted 28 March 2012 - 08:39 AM

sometimes "skins" are left behind in the wells (caused by loose fitting combs). these skins will prevent the sample from reaching the bottom of the well. they can be scraped out with a thin spatula or sucked out with a syringe needle connected to vacuum, be careful to not damage the gel.
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#5 pobrien

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Posted 29 March 2012 - 08:02 AM

Problem solved. After troubleshooting the loading buffer, gels etc I found out that I 10X running buffer was used instead of 1x. ...very embarrassing Posted Image




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