A method for analyzing or purifying samples. Molecules are separated by their relative weight, charge, or stereochemistry, which affect their speed of movement through a gel.

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6
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1answer
327 views

Does GTP-γS (GTP gamma S) bind all GTP-binding proteins?

I've just read an article Rab10 GTPase regulates ER dynamics and morphology - Nature Cell Biology 15, 169–178 (2013) doi:10.1038/ncb2647. In this paper, to identify Rab proteins in ER, first they ...
2
votes
1answer
659 views

Where does Taq polymerase migrate during electrophoresis?

Assuming a 1% Agarose gel with TAE. Follow up questions: Is there a way to stain for it? Could the polymerase be captured and reused?
10
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1answer
121 views

When running a gel, what could cause a standard to run “faster” than usual?

We are running a gel on the products of a restriction digest to determine the size of an insert. We know the vector backbone size should be ~2.9kb; however, the standard appears to be running too ...
12
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1answer
5k views

When running gels what is the difference between constant volts or constant amps?

In general, you want to be consistent with running your gels either at constant volts or constant amps. However, it is very clear that during the progression of both PAGE and agarose gels, the free ...
7
votes
2answers
6k views

How do I deal with sticky and viscous samples from cell lysates?

To check a protein expression I pelleted a small amount of E. coli before and after induction and lysed them by redissolving them in SDS-PAGE loading buffer and heating them to 95 °C for 1 minute. ...
3
votes
1answer
264 views

What is the DNA/protein charge ratio?

To study DNA-protein interaction, I want to do a DNA retardation test by mixing the protein with DNA and afterwarts loading it on an agarose gel to see if the DNA migrates slower. I've found some ...
1
vote
1answer
2k views

Is there a free alternative to Gelcompar for comparing banding patterns across multiple gels?

In order to run my microbial community samples from my experiments through DGGE, I was required to use multiple gels. Thus it is necessary to compare banding patterns across more than one gel. ...
12
votes
3answers
15k views

How many agarose gel bands are typical for circularised DNA

I am aware that circular DNA can be both relaxed and super coiled. However when running an agarose gel of the circular plasmid along with singly digested plasmid with BamHI and HindIII, I see 1 band ...
7
votes
1answer
5k views

How do Proteins migrate in MES vs. MOPS

My gels look significantly different in MES (2-(N-morpholino)ethanesulfonic acid) and MOPS (3-(N-morpholino)propanesulfonic acid). That is to be expected. What I don't understand is why the simple ...
5
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1answer
10k views

How do SDS-PAGE gels differ in a Bis-Tris system vs. a Tris-Glycine system?

Protein migrate differently in Bis-Tris and Tris-Glycine gels. I was curious about the actual reasons why. Do certain gel systems result in a tighter resolution than others?
11
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1answer
1k views

Alternatives to TBE buffer for denaturing polyacrylamide gel electrophoresis of DNA and RNA?

I just stumbled upon an article promoting 10 mM sodium borate as an alternative to the well-known TAE and TBE buffers for agarose gel electrophoresis of DNA (Brody & Kern, 2004). They claim that ...
6
votes
1answer
7k views

What would cause bands to appear lower on a nonreduced SDS-PAGE gel?

I have a protein gel and I'm comparing the reduced with the non-reduced gel. On the reduced gel, I have a few shorter bands but these bands appear with low intensity. In the non-reduced gel, I'm ...
3
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1answer
755 views

How often can you reuse coomassie stain?

I usually heat to boil in the microwave, and stain for 12 minutes. How often can I reuse this buffer?
4
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1answer
97 views

Is there a cheap and easy way to check for potentially unsafe UV exposure?

My lab has an older large Fotodyne tabletop UV transilluminator for visualizing gels. It has a UV blocking cover that hopefully reduces UV that passes through, but I've always been suspcious as to ...
11
votes
2answers
7k views

Why should I degas my gel solution for polyacrylamide gels?

In protocols for polyacrylamide gel electrophoresis (PAGE) I often see instructions to degas the gel solution by putting it under vacuum for 10-15 minutes before polymerizing the gel. I usually ...
8
votes
2answers
2k views

Alternatives to ethidium bromide for staining small nucleic acids?

While ethidium bromide works well for staining larger single-stranded RNA or double-stranded DNA molecules, it doesn't stain smaller nucleic acids very well. I observed that at around 20 bases and ...