dialysis tubes for chromatin purification - (Apr/19/2007 )
Hello!
I want to isolate native chromatin from heart tissue and im using some suitable protocol. According to this protocol i need to fractionate the chromatin with MNase . The first soluble chromatin fraction comprises small fragments only ((one/two nucleosomes), after that i have to recover a second chromatin fraction (comprising larger fragments of chromatin:penta, tetra,tri) by overnight DIALYSIS.
The problem is that im not sure what are the characteristics of the dialysis membrane/tubes i have to use (its doesnt mentioned in the protocol)...should i use membrane with molecular weight cutoff 6-8kDA or 12-14 kDA? or even 3.5 kDA? i think it have to be 6-8..but im really no sure...
Thank you!
Did the protocol mention what does this step do? Removing small ions, or detergent? If it is for getting rid of small ions, then anyone will work for you. 10k-12k is the cheapest.
Thank you!
The purpose of this step is to remove small (one or two nucleosomes) fragments of the chromatin (DNA+histones) and to keep only the long chromatin fragments (three, four and five nucleosomes long)...
By what mechanism? Size difference/dialysis or selective precipitation for large ones?
If it is former, you need to calculate the MW of monomer, which will be ~200bpx660 + ~8X15,000= 252,000 d. You need a dialysis bag that has a MWCO 250,000 to 500,000 to remove the monomers. However, dialyzing away a gaint particles like this will take a very long time and may not be practical to do so in comparison to spearation using a size exclusion column method.
If it is the later, whats the trigger mechanism for precipitation of larger nucleosomes?
More info is need to make a call on that.
If it is former, you need to calculate the MW of monomer, which will be ~200bpx660 + ~8X15,000= 252,000 d. You need a dialysis bag that has a MWCO 250,000 to 500,000 to remove the monomers. However, dialyzing away a gaint particles like this will take a very long time and may not be practical to do so in comparison to spearation using a size exclusion column method.
If it is the later, whats the trigger mechanism for precipitation of larger nucleosomes?
More info is need to make a call on that.
Thank you for answering me! You are helping me a lot with the understanding of the method!
I think its about the size difference/dialysis.
A little background: I want to purify native chromatin in order to perform chromatin immunoprecipitation (Chip) for histone acetylation analysis. The optimal chromatin fargments size for immunoprecipitaion with the specific antibody is 200-1000 bp.
In order to perform the chromatin fragmentation im putting my isolated chromatin in buffer wuth MNase (microccocal nuclease)
after this digestion step my goal is to purify the 1-5 ucleosomes fragments of the chromatin.
So firstable, according to the protocol, im duing centrifugation to the digested chromatin and my supernatant comprises from small fragments only (one-two nuleosomes long) - fraction 1.
After that i have to submerge the pellet to 12-16 hours of DYALISIS. The centrifugation after the dyalisis step recovers the SECOND soluble chromatin fraction (in the supernatant), comprising larger fragments of chromatin (3-5 nucleosomes), the larger than 5 nucleosomes fraction stays in the pellet.
Now, i have to mix the two factions (the 1-2 fragments+ the 3-5 fragments) in order to get the optimal size of the chromatin fractions for the next steps.
I cant really understand why i have to do the dyalisis step ...cant i just centrifugate after the MNase digestion in order to recover 1-5 nucleosome fragments? what exactly im ridding off by the dialysis? from the smaller than 3 nucleosomes fragments? if it so, what MWCO dyalisis bags you are recommending me?
THANK YOU VERY MUCH one more time!!!
Hi!
I think im finally understand the purpose of the dialysis step!!!
Its not suppose to separate the chromatin fragments (centrifugation process doing it)! The dialysis only suppose to clean the left overs after the first centrifugation in order to recover more 1-5 nucleosome fragments...you were right suggesting that dyalisis is used for removing small ions and detergents that may be contributing to the small chromatin fragments shielding (?) and i have to recover it... so, its ok to to use 12-14 kDA mwco dialysis membrane to do it?
Thanks a lot! You were really helpful!!!