0
$\begingroup$

For an experiment, I am trying to fix the mononucleosomes (100ng) using formaldehyde as crosslinking agent in HEPES buffer. I have been using 2% formaldehyde in a reaction buffer containing 1mM EDTA, 25mM NaCl and 50mM HEPES, pH 7.5 (same as nucleosomes suspension buffer) to fix mononucleosomes.

In my first reaction, I added 100ng of nucleosomes, 2% formaldehyde and incubated at 4degC, 30', then I added 0.4% SDS and incubated for 30' at RT. I observed less intense but equally sharp band for nucleosomes (less smear) as compared to unfixed nucleosomes.

When I performed the same reaction again, the gel showed a huge smear covering the entire lane instead of a sharp band.

I have been reading about the fixation reactions and encountered an emphasis on using methanol free formaldehyde (while I was using 10-15% methanol containing formaldehyde). Do you think that can be one factor for not being able to get similar results?

$\endgroup$
  • $\begingroup$ Same reaction protocol giving undersirable result may simply be because of some sample prep errors. See this protocol. The final concentration of formaldehyde is 1%. Most protocols suggest that formaldehyde be prepared fresh. This is because formaldehyde gets oxidized to formic acid. $\endgroup$ – WYSIWYG Jun 24 at 8:51
  • $\begingroup$ Thanks for the suggestion. The chance of adding errors while making the reaction is relatively constant since I prepare it in the same manner, every time (except for the pipetting errors). I agree with you about the oxidation of formaldehyde. The protocol you suggested uses 11% of final formaldehyde, is it because they are using certain mammalian cell lines? $\endgroup$ – Jyoti Sharma Jun 24 at 12:03
  • $\begingroup$ Their fixation buffer contains 11% formaldehyde. They add 20ml of this buffer to 200ml of cell suspension. So final concentration of formaldehyde is 1%. $\endgroup$ – WYSIWYG Jun 24 at 12:08
  • $\begingroup$ Yeah, I figured. Thanks though. $\endgroup$ – Jyoti Sharma Jun 25 at 8:09
  • $\begingroup$ Since formaldehyde is the matter of discussion here, I wanted to ask that I have an 1ml aliquot of pure formaldehyde sealed with inert gas in a glass. Now, in one reaction I am only using 10ul from that 1ml. Ideally since these aliquots are for one time use, I shoud discard remaining 990ul but if I want to store, is there a feasible way to preserve the rest of the formaldehyde while taking care of its exposure to air? $\endgroup$ – Jyoti Sharma Jun 25 at 8:15

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Browse other questions tagged or ask your own question.