NP-40 Nuclear Lysis Problem - (Apr/23/2011 )

Apologies if this in the wrong section

I have been lysing nuclear proteins from my cells using 2 buffers, the first buffer is added to the whole cell pellet and shaken for 10 minutes. Then I add 1ul (yes 1ul!) of NP-40 to each samples, shake for further 5 minutes, spin, then recover the supernatant (cytosolic fraction) and then proceed to lyse the nuclear pellet with the second buffer.

The problem I have had lately is I think there is too much NP-40 getting in my cell lysate. As a result, the nuclear pellet is opaque/glassy and globby (hard to resuspend) as opposed to the usual white pellet. Further still, when I come to do Bradfords on them, the colour goes bright blue and there is serious overestimation of protein concentration.

First question, is the intermediate step of adding 1ul NP40 necessary for lysis? I understand the detergent is designed to rupture the cytosolic membrane, but the first buffer does not contain any detergents. The second buffer used to lyse the nuclei DOES contain NP-40.

Second, is there a way to recover the contaminated samples by removing the detergent so the samples can be used for analysis?

I usually nail this lysis procedure perfectly and the fact it recently happened in only 2 of 6 samples suggests to me I accidentally added more NP-40 to the lysates.

Thanks

Dave

the gooey thing is the genomic DNA. you just need to add 1 ul of DNase. any concentration would do. sonication doesn't work most of the time for me. I think I've posted this more than 100 times at this forum
btw, most people use sodium deoxycholate to break down nuclear membrane.

your first buffer must be hypotonic buffer. there are various methods for cellular fractionation. I used digitonin method. it's a milder detergent. and I never used Dounce homogenizer. I hate it.

also, getting the gooey pellet is really random, in some sample it happens, in some it doesn't.

Curtis on Sun Apr 24 12:08:06 2011 said:

the gooey thing is the genomic DNA. you just need to add 1 ul of DNase. any concentration would do. sonication doesn't work most of the time for me. I think I've posted this more than 100 times at this forum
btw, most people use sodium deoxycholate to break down nuclear membrane.

your first buffer must be hypotonic buffer. there are various methods for cellular fractionation. I used digitonin method. it's a milder detergent. and I never used Dounce homogenizer. I hate it.

also, getting the gooey pellet is really random, in some sample it happens, in some it doesn't.