So a few weeks ago I started running cell fractionations in a 15% polyacrylamide gel that I prepared about an hour prior to running. I run all my gels at 20mA per gel and since I only ran 1 I just used 20mA. Typically, these gels take about 1.5 hours to run at 10% or 12% but for some reason it was done in under an hour. The blue dye in the laemmli buffer looked odd as well as normally they kind of mush into a single looking line with just little blips for each well but the dye never fully flattened within the well and looked like a smear just moving down the entire time. I attached an example of what I mean. The first is the fractionations and the second is a test I did with whole cell lysates. The first time I just thought I made a mistake in the gel prep but it happened 3 times now with the cytosolic and nuclear fractionations and now again with my whole cell protein extractions. My thought before was the high salt in the buffer for cell fractionations was messing with the running step but now I don't know anymore as the cell lysates are just in RIPA which I've run a hundred times with no issue.
It seems I am the only one having this issue as my lab mates have run multiple gels with no issues. The only difference is they were using 10% gels but I don't see why my higher percentage gel would run faster or different as it never has before. My voltages are getting higher than normal as well progressing up to about 190V within 30mins at 20mA.
Has anyone ever had this problem or seen gels run in this manner because I am at my wits end with this...
All my buffers, gels, and samples preps are made fresh and my lab mates are using the same reagents and buffers as me but aren't having this issue.
Not unfamiliar with this kind of runs, and it's always hard to find out of it. I've been running 6 to 15% gels, human whole cell lysates, cell pellets, saliva, serum, bacterial lysates. I guess it is due to differences in content between the samples. Since a 15% gel is more compact, larger molecules will have greater difficulties running through it, and if there are much more of these in one sample, it can run a bit slower and squeeze the neighbor well.
I start running the first 10-20 min at approx 10mA so the sample is focused at the border between stacking and running gel, and then run at 200V, so it is finished within approx 1 h. Tried cooling down the buffer and so on, but found out that equal load is the most important part.
Part of the dye always runs faster, but the front of the proteins will be in the dark blue line on the picture 142323. Although it looks like this while running, as long as the gel is homogenous, the same protein will probably be at similar height, even in "squeezed" lanes. I ran last week with ladder in 3 of 15 wells (that will be a nice test of the gel), where border wells did not run as far as the middle one. The final difference was only found in the last 5-10mm.
Looks like the stacking process (isotachyphoresis) is not working. Generally, there can be three reasons:
- the gel was wrongly prepared (like mixing up stacking and separating gel buffer)
- the sample contains a lot of salt, that screws up the stacking process.
- there is a lot of DNA in the sample, which increases viscosity and may interfere with electrophoresis
The solution to the first problem is obvious, for the second problem the easiest solution is a methanol/chloroform precipitation of the protein (doi:10.1016/0003-2697(84)90782-6). For the third problem, sonication may help, or passing the sample through a 27G needle a couple of times.
I'd also recommend 10 mA for a minigel in the stacking phase, and 20 mA once the front has entered the separating gel. If your system has a cooling loop, use it to prevent heating as much as possible.
I'm with Mr. Buxbaum. Is your first time running 15% gels? It looks like you are pouring your stacking solution slowly and it "polymerizes" before you put it into glasses.
Daniel Belcher ,
What do the gels look like when you stain them? Any difference from "normal" gels?
Engelbert Buxbaum My thought was the salt concentration as well but I have had other in my lab do the same procedure and their EP ran fine. I am going to try diluting the preps a bit to see if that helps as well as the method you posted thanks.
I do not believe it is the gel as this has happened on 3 separate occasions with 3 freshly made gels.
Alejandro Martin I am getting additional bands and some non-specific binding that I have never seen before in my previous uses with Histone 3 and my tubulin blot showed that the protein didn't even fully migrate through the gel as it was at too high of a mw a dispersed across multiple lanes. H3 was able to fully migrate but again, had additional bands that I have not seen before nor should be there.
Hey, can you tell me which protocol are you using for fractionation? Thanks
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