Hi to all,
I have performed several experiments to study the interaction between a protein and a DNA strand supposed to be its binding site.
I've studied this interaction with the recombinant protein and that DNA probe by mean of various techniques such as in solution NMR, Crystallography, Native PAA gel Electrophoresis, EMSA Assay, but I have never seen any interaction.
However by means of EMSA Assays performed on nuclear extracts, and the DNA probe, i See the interaction.
Recently I tried to perform a pull down assay with the streptavidin coated magnetic beads: I have performed a control experiment with recombinant protein and biotin (without any Biotinilated DNA) in order to test the specificity, an experiment with nucler extracts, and an experiment with the recombinant protein.
Furthermore Solutions and buffers I used, are the same used in the EMSA assays and so on.
Once I eluted the protein from the beads by means of denaturing and reducing conditions, I performed an SDS page followed by an immunoblot.
I see that the DNA probe is needed to elute the protein. the protein is interestingly in a dimeric form. the interaction seems to be specific, because signals in the control sample without DNA are very weak. this can be observed both on nuclear extracts and on recombinant protein.
what does it means?
Why I can see an interaction between the recombinant protein and DNA probe with Pull down and not with EMSA?
How Can I Isolate the alleged Protein Dimer and characterize it by means of other techniques such as MS or NMR?
so is there another method i can use to elute the complex that avoid the use of denaturants os salts?
Thank you
If I understand you correctly, you were able to pull down the protein using streptavidin beads attached to biotinylated oligonucleotide probe, and you found the protein was a dimer. You didn't say how you determined the protein was a dimer (not, presumably, by SDS-PAGE, unless you chemically crosslinked it first).
You are worried because the other techniques you tried showed no binding. It may be that the binding affinity is relatively weak and you were able to see it by pulldown because the concentration of everything was high enough to drive the equilibrium towards binding.
One way you could try to measure the binding affinity quantitatively is to prepare the oligonucleotide probe with a fluorescent dye attached (fluorescein is the cheapest if you are going to have it synthesized) and use fluorescence polarization. As you increase the protein concentration at a fixed oligo concentration (e.g. 100 nM), the fluorescence polarization increases as the proportion of oligo bound to the protein increases.
The nice thing about this experiment, if you have access to a plate reader capable of fluorescence polarization measurements, is that you can do it with very little material by working in small volumes, such as 10 microliters/well (using low-volume 384-well black microplates). Every protein concentration should be done at least in triplicate, and controls should be made without the fluorescent oligo. You should demonstrate that you can compete the binding with the same oligo lacking the fluorescent dye, to make sure the protein isn't recognizing it.
Thank you for the very interesting advice! The odd thing in this experiment is that the protein elutes from the beads as a dimer, having a weight double respect to the one of the monomeric form. Furthermore, it is very unusual that I have a diner although the denaturing and reducing conditions. I can't explain this.
could it bee that you see an non direct protein dna interaction? this would explain why recombinant protein does not interact
perhaps I got your points wrong, but it could be possible, that your portein of interest is not alone capable of binding DNA. So this could mean, that it binds another protein which moderates the binding or which is the DNA binding component of the complex
ahh ok, now i got you!
I don't think that there is a tripartite complex, because while in the EMSA Assay only the protein within the nuclear extracts interacts, in the pull down assay both the recombinant and the protein in the nuclear extracts are pulled down by the beads.
Furthermore the weight on the SDS-PAGE of the eluted fractions are consistent with a dimer of my protein, and I have the same result both with the extracted protein and with the recombinant one.
I wonder how u prepare your western samples, because without crosslinnking oligomer should not be detected. Do you crosslink your proteins before separation?
No, i don't. I simple elute them from the streptavidin beads in lemmli Buffer at 95°C, then i precipitate the beads with a magnet, eventually I collect the supernatant and I load it on a PAA gel. After the electrophoresis I transfer the gel on a pvdf membrane, and So on
So are u sure to detect a dimer? At least i would guess laemmli is disrupting PPI. So you should not see dimers
Hi to all !
I have the same problem in my DNA pull-down experiences. After the DNA pull-down, I detects the protein with a larger size than the expected size (approximately 20 kDa more) in a SDS-PAGE. I can't explain this.
Have you found an answer to your problem since March 2015 ?
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