triton x and np40 for nuclei isolation - (Mar/02/2005 )
I'm currently in the beginning stages of nuclear run on preparations.
For the isolation of the cell nuclei (MCF-7 and MDA-MB-231), the recipe for the lysis buffer is 10mM Tris pH 7.5-8.0, 0.5% NP-40, 10mM NaCl, 3mM MgCl2.
Problem is, we don't have NP-40.... we do have triton X.
I've been googling a bit, but, still would like to have someones opinion on whether these two are interchangable.
Really don't want to screw things up, anymore than what is destined to happen anyway.
Can I use triton X? How much?
Thanks a million in advance.
Hi, vetticus3. I've never done run on, but I've done a lot of NE for EMSAs, ChIPs of WBs, and always have used NP40.
According to the Sigma product info sheet:
In my experience, used at the same concentrations, NP-40 (or Igepal), is more stringent tant TX100. So if you have no option but to use TX100 I would tell you to use 2-3 times the amount of NP40 in your protocol... But I strongly recommend you get some Igepal.
I have in the past used Triton-X100 in exchange for NP-40 with no differences in results.
I used to do cell permeabilisations for confocal studies and WB and found that in the presence of 0.3%-0.5% Triton-X100 the nuclear membrane would remain intact however the cytosolic membrane would be permeabilised.
Consequently, by lysing the cytosolic membrane, spinning and then using a nuclear solubilisation buffer usually high salt or the addition of CHAPS or SDS then you could obtain clean nuclear lysates.
Hope this could be of some help
Triton X-100, Igepal CA-630 and NP-40 are non-ionic detergents and may be used interchangeably. All have 275 nm absorbance.
Ethylphenolpoly(ethyleneglycolether)x or polyoxyethylene(10)isooctylphenyl ether or t-Octylphenoxypolyethoxyethanol or polyethylene glycol tert-octylphenyl ether (x is approximately 9.5 according to the Sigma catalog) is a non-ionic detergent. polyethylene glycol P-1,1,3,3-tetramethylbutylphenyl ether, octyl phenol ethoxylate, 4-octylphenol polyethoxylate, Mono 30
Triton X-100 is an estrogenic surfactant spermatocide, closely related to Nonoxynol-9. Octylphenol exhibits estrogenic effects in some animals at 30 ppb which is roughly 0.000009% Triton X-100 or one drop in 275 liters of water.
D = 1.070 4-(C8H17)C6H4(OCH2CH2)nOH CAS 9002-93-1
Strictly speaking, Tergitol NP-40 (Union Carbide) previously available as a solid at room temperature as Sigma catalog number NP-40 is not the same as Nonidet-P40 which is a liquid at room temperature ("Nonidet" is a registered trademark owned by Shell Chemicals).
Tergitol type NP-40, (a solid at room temp.) and Nonidet P-40 (no longer available but Sigma sells its equivalent, Igepal CA-630, a liquid at RT).
A recent Fluka catalogue lists 'Nonidet P-40 substitute' with synonyms being NP-40 (as unfortunately Nonidet P-40 was sometimes called) as well as Imbentin-N/52.
Sigma Cat # I3021 IGEPAL CA-630 (octylphenoxy)polyethoxyethanol
“chemically indistinguishable from Nonidet P-40, which is no longer commercially available”