I´m trying to do a DNA/Protein Gel Shift Assay using agarose gel to check if my protein is binding DNA or not. The problem is the PI of my protein is high (around 9.5) so the sample won't run properly on the agarose gel. I tried to slightly increase the pH of the buffer used to run the gel but still no improvement. I was thinking about decreasing the pH of the buffer used to do and run the gel + inverting the running position to run from positive to negative but in this case I can't run a DNA sample alone in order to see a shift in the bands.
Anyone ever tried this? And any solution for such problem? Thank you in advance.
Dear Nada,
it is quite common that DNA-binding proteins have very high PI, so that the interaction with the DNA (negatively-charged) is fostered.
Actually, I have worked on a protein with a similar value of PI and carried out band-shift assays both in agarose and polyacrylamide gels without any problems.
What do you mean when saying that the samples won't run properly?
At the end of the experiment, you should follow the migration of the DNA molecules that will run for sure in the agarose gel. It does matter if the protein does not migrate alone in the gel...it will be drag from the DNA or viceversa (it depends on the point of view).
Anyway, if your protein interact with the DNA, you will see that the DNA-band will be shifted up...in any case you are not going to visualize the protein band, unless you stain the gel with a protein dye.
By the way, I agree with the suggestion of Sanjiv Kumar of running a polyacrylamide gel.
About the concentration of the acrylamide, it mostly depends on the size of the DNA-probe you are using.
I agree with Salvatore, you will see if the DNA band shifts or not. But of course it's not very elegant and my experience is that the bands are often very smeary for DNA-Protein complexes where the protein shows high PIs.
You could try MST (MicroScale Thermophoresis) instead. It does not require any gel or matrix, but you can quantify interactions in free solution in a fast, easy and precise way.
http://www.nanotemper-technologies.com/news-events/news/article/microscale-thermophoresis-more-than-binding-affinities/
Hi Salvatore, Of course i can see the DNA band alone as a control on the gel but when im running the protein sample that is incubated with the DNA the band is stuck up and i can´t see any band down on the gel! its either stuck up where i loaded my samply or i would see a band on the opposite side of the gel ! and i don´t know what does that mean !
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