Hi,
I am working on DNA and protein crystallization. Since my protein have homotetramer form and stabile in high salt concentration, it was make problem to me to binding with DNA. I would like to crosslinking both of them by crosslinker.
Does anyone have a journal of experience about that?
Thank you in advance.
From the Thermo-Fisher web site:
"Crosslinking of DNA or RNA to proteins is often difficult because the reactivities of most crosslinkers favor protein-protein crosslinking over protein-DNA crosslinking. To assist in these crosslinking methods, DNA probes are often synthesized with primary amines or thiols attached to specific bases. After insertion of the bases into DNA, amine- or sulfhydryl-reactive crosslinkers can be used for their conjugation to proteins."
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/crosslinking-applications.html
Here is a paper on DNA-protein crosslinking by formaldehyde and methylene blue:
http://nar.oxfordjournals.org/content/28/2/e4.full
And here is a whole book on the subject:
http://www.ncbi.nlm.nih.gov/books/NBK7107/
Hi Shapiro, thank you for your information. In my opinion, your paper tells about in vivo cross-linker.
I have purified bacterial protein and I thought about in vitro-cross link with oligonucleotide (DNA). Do you have such kind an experience?.
Many thanks
Sure honey, I've done the the expt so many times , I'm a mol biologist. What Adam says is absolutely correct, I'll just add a modification, i work on cancer but still i'm acknowledged in a paper in bba journal for this work. 1> choose your DNA carefully, not arbitrarily.DNA protein cross linking causes aging so think of something that accelerates DNA-Your-Protein cross linking.2>Warm the solution gently 50 to 60'C, (and add beta-SH and EDTA ) for half an hr. What do u want to do?keep the complex in solution or extract the dna or show Dna-amino acid complexes?> For the last do an ELISA
I think you should opt for complementary DNA , that can encapsulate your protein.
in order to acheive this, you have to screen double stranded oligonucleotides binding capacity to your functional protein.
then chose a concentration w to stabilise complex,
and you can visualize your complex.
Hi Mukherjee and Reddy,
Thanks for your suggestion. I am crystallizing protein-DNA complex. But, I facing problem. My protein is homotetramer form and stabile in high salt concentration. In this situation is difficult to bind with DNA. I was tested several kind of DNA such us length and overhang sequence. I obtained so many crystals in several crystallization conditions. But, after X-ray diffraction and refinement, I could not observe the DNA density. Probably, DNA was not bind into protein. So, that's why, I would like to use linker to maintain these complex. Do you have some idea about what kind a linker?.
Many thanks
There is a method in our group for crosslinking, you can check it from this link.
http://www.nature.com/articles/ncomms6849
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