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?