Dear all,
I have been trying to run my samples (cellular lysates) on an SDS-agarose gel (low melting). The first try outs were terrible, but I gain some knowledge about my limitations (like pre-warming the glass plates before I add the low-melting agarose solution). I am having now the challenge of removing the combs without tearing in half the gel. I have the impression that my problem relies in how thin the gel is (0,75 mm). I have been thinking about using a 1,5 mm thick vertical system instead. Are there any other tricks that you can recommend me?
Thanks for your help,
Luisa
Might be my ignorance, but why are you using agarose when you could use acrylamide...? ie SDS-PAGE
Hi Stephen, that is coming to the same point I would like to address: How big is the protein she wants to look for since you can get already gradient SDS-PAGE 4-12% for example?
You can try coting the comb with Repel-silane prior to the assembly should solve your problem. But still in my opinion also to resolve most of the proteins acrylamide gels should be fine.
Sorry.. I just saw all your messages...
The protein is between 270 and 350 kDa, with different isoforms. I decided to try agarose, because low porcentage acrylamide gels loose the resolution of the bands. (I have tried from 5% to 8% letting run out a lot!)
And, I suspent it is being modified, so I really need an excellent resolution of the bands to notice small changes in the running pattern. For some actin-related proteins have been publish the agarose gels as ideal for differentiation of modified proteins... that is the reason...
to Dr. Mpidevi: What exactly is the repel-silane?
Thank you all for your comments,
Luisa
I found the repel-silane!, but since my combs are not glass, will they still work?
repel silane probably wont do anything with your teflon combs. You could try wiping a very thin layer of petroleum jelly on the comb to produce the same effect. You could also use a combined agarose/acrylamide gel. The agarose basically provides the firmness while the actual seiving effect is due to the polyacrylamide network. This allows you to use much less than 4% polyacrylamide, even down to 1% or less for bigger proteins.
Dear Nick, what exact proportion agarose:acrylamide should I use? I assume, I should keep using the low melting agarose...
Thanks
I would start with something like 1% agarose, 2% acrylamide and see how it performs. If your proteins move too fast without decent resolution go to 2.5 or 3% acrylamide.
just did a quick google for agarose-acrylamide gels and came up with these references which may help
http://www.jlr.org/content/27/4/457.full.pdf+html
http://www.sciencedirect.com/science/article/pii/0003269782904894
http://onlinelibrary.wiley.com/doi/10.1002/elps.1150111020/abstract?syshttp://onlinelibrary.wiley.com/doi/10.1002/elps.11501601221/abstract?
Dear Nick: THANKS A LOT!!! I will get the references up and running and I will try to keep here an update of the trials...
I only looked for Agarose gels, without the mix...
Another question: I have found (for both kind of gels) that adding glycerol to the mix will increase the definition of the bands... have you had any experience with that?
sorry Luisa, its actually been a number of years since I was playing with that stuff and all the important details one accumulates from doing something every day are long forgotten
Don't worry... I know how it is... I think I will give it a try... Thanks a lot for your help!
Hi Luisa...
Nice to have a comrades in arm about this agarose gel. I'm doing the same thing with a protein size around 4000 KDa. I had problem similar to what you said and managed to make gel up to 2.5%. 3% and above is still an epic failure. About the comb, if we make the end of the teeth a little bit roundish, just cut the tip of the teeth a bit and it will come out without any problem. I don't know if your still doing this after a year though. And this paper about the glycerol
http://www.ncbi.nlm.nih.gov/pubmed/12783444
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