Hi, I have problems with running gel electrophoresis. I have tried agarose gel electrophoresis and native PAGE.
I have two proteins, which have molecular weights of ~30kDa and ~180kDa
and two aptamers (oligonucleotide), one is 51nt and the other is 18nt
I have to check whether they bind: ~30kDa with 51nt, and ~180kDa with 18nt
So, what I did was put different amounts of protein (10-80 pmol) with 10 pmol of the corresponding aptamer and fill the volume with nuclease-free water. Incubate the samples for >1 hr and do gel electrophoresis.
Under Bio-Rad Chem Doc, I see bands at the top and the bottom of the gel (for both samples).
First, did electrophoresis with 1.5% agarose gel and I saw no binding.
Then, I tried native PAGE with different pH buffers and that did the same thing as the agarose.
These aptamers do bind with corresponding proteins because numerous research articles have used the same sequences.
Please help. What should I do now?
Dear Susan,
are you sure that you don't need any binding buffer when you prepare your chimeric aptamers? I'm not an expert of chimeric aptamers composed by aptamer and proteins, but I work with RNA/RNA or RNA/DNA chimeric aptamers and I use a binding buffer when I prepare the chimera.
I agree with Lorena, binding the aptamers to the proteins should be done in a salt containing buffer.
I am a little confused by your statement: "Under Bio-Rad Chem Doc, I see bands at the top and the bottom of the gel (for both samples)." You said that you didn't see binding in the agarose or acrylamide gels immediately after this. If your experiment works, you should expect to see two bands at the top and bottom of the agarose gel (lower will be free aptamer, upper will be bound aptamer).
Lorena Quirico, do you use binding buffer for the aptamers (nucleic acids) only? or for proteins as well?
John Hardy Lockhart, I saw two bands because protein and aptamer did not bind. I ran gel electrophoresis separately: one for aptamer 1 and protein 1, and the other for aptamer 2 and protein 2!
How are you visualizing your proteins/aptamers? (e.g. fluorophores on the aptamers?)
Doing an electrophoretic mobility shift assay in the agarose gel will only produce a shift in the aptamers band given the huge size of proteins in relation to the aptamers. This can produce two bands if there is excess aptamers that do not bind to the protein.
You'll need to run lanes with the aptamers alone and the protein alone for comparison.
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