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several questions on running SDS-PAGE


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#1 southpole

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Posted 25 April 2012 - 07:23 AM

Hi, I'm struggling with running my protein samples on SDS-PAGE.

I have some several questions and my thoughts on these problems.

1. My samples are 'sticky' before loading on the gel.

I added 6XSDS buffer into my lysate sample and incubated in 37℃ for 30 minutes(the protein I want to see is a membrane protein, so I can't boil it, or it'll aggregate w/ its hydrophobic regions)

and when I started to load it in the well, it won't fall off like a string, but it rather clumps down together slowly and doesn't fall off easily. If I try to pull the pipette away, the sample drags out and streches away.

At first I thought it was b/c of the gDNA, but I centrifuged before and took out the supernatant for the lysate sample. However, I might have to check exactly at which step it starts to get sticky.

I'm suspecting that the sample might be less denatured. I ran the SDS-PAGE of the same protein before and it was also sticky when I loaded that sample. The band did not appear in the right position but in a higher position, and I think this was b/c the protein is denatured, it might have been hard for it to pass thru the gel than when it is linearized and denatured.

2. How does the pH of the resolving gel affect the running?

[font="돋움, dotum, sans-serif"]I think if the pH of the resolving gel is over 8.8(the pH of the resolving buffer, Tris-Cl, is 8.8), then there would be more negatively charged glycines and under the same voltage, or current, the proteins would receive the less current, thus less power to move thru the gel, and it would take them longer, especially the bigger sized proteins, to migrate thru the gel.[/font]

[font=&#46027]Thank u for reading, and how do you think about these problems?[/font]

[font=&#46027]I'd appreciate your thoughts.[/font]

[font=&#46027]Have a nice day.[/font]



#2 bob1

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Posted 25 April 2012 - 04:48 PM

Centrifugation will not necessarily remove the DNA, you could try sonicating or passing the solution through a fine gauge hypodermic needle several time to shear the DNA.




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