I'm doing 2-dimensional gel electrophoresis and want to run two samples next to my strip for comparison: marker (so check size) and the rehydrated protein (for comparison to the 2D spots).
I know it's not optimal, but would it work to run a protein sample and the marker in the same (only) well on the gel? Some of my proteins are the same size as the standards on the marker, but the concentrations would be quite low.
I'm visualizing the samples with ECL and it's a prestained marker.
Thanks,
Lauren
Are u saying for 2nd dimension? You can, make two small wells at one end, and run those samples and your strip/tube gel after that.
Yes, for the second dimension.
Is there an easy way to make an additional small well/split the pre-made well in a precast gel?
I used to cut the comb to single well size (if you have spare comb), and put them either on one side or one at each end. You need very small size well, so don't keep it too deep. The less the size and less volume of your samples, the better it will run along and give you correct pattern.
I'm actually using pre-made gels (I have enough sample-to-sample variation I don't want to add any more by pouring gels...).
By that logic, do you think it would work to remove the comb from a premade gel, cut a slit in the comb so that the one well would be two smaller wells (leaving the IPG strip well as is), and then reinserting the comb into the gel and filling the cervice with a little bit of agarose?
Ohh..I have never used precast gels. what precast gels are you using (company/type/%)? You can try what you are saying by just running one or two different markers/some spare sample, don't waste your exptl. sample here. If it looks fine, then try with samples.
I'm using Bio-Rad Criterion Tris-HCL 10% polyacrylamide gels. Unfortunately these gels only see to come with the option of IPG strip + 1 well. Hence why I was thinking to try running the marker and my sample in the single well...I'll have to give it some more thought and some trial runs I guess
Then I suggest you run sample and marker in one set and just marker in other, so that you can distinguish between the bands. Later on, you can easily superimpose them and use the mixture profile for your gels.
Yes, you can mix them and load into the same well. It's not a big deal.
i always load my marker on a paper wick and just drop the paper wick in the corner of the gel.You could load try and see if this works for you. I have never tried loading protein samples this way but i know it works for the marker. Good luck :)
No problem to mix it. To make it easier: use coloured markers. They are available from a lot of supplieres.
my marker is prestained, so I think I'll give it a shot in the near future. Will let you know how it goes!
Also, @Maggy, how do you do that? Do just soak the wick in marker and stick it at the side?
Thanks all!
Actually I usually cut the wicks into half and use one 5ul on on one half.Make sure you put the paper wick on the gel before u add agarose in case you are using agarose as your dye front
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