Sample does not sit in the well despite (glycerol checked)
Posted 27 March 2012 - 09:50 AM
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.
Posted 27 March 2012 - 11:32 AM
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.
Posted 27 March 2012 - 12:28 PM
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.
Posted 28 March 2012 - 08:39 AM
genius does what it must
i do what i get paid to do
Posted 29 March 2012 - 08:02 AM