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Need help for prepping western samples with NP-40

NP-40 NP40 western western blot

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#1 Druss00

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Posted 09 August 2012 - 09:01 PM

Hello,

I am new to western blots and have a question about prepping my tissue culture samples (293T cells). After pelleting my cells and removing PBS (just to wash them) I add around 100uL of NP-40 to my pellet and re-suspend it. After letting it sit on ice for 30 min (vortex every 10 min) I spin it down at the highest speed our centrifuge will go (this is all done in 1.6mL "epi" tubes) to pellet the nuclear DNA. The problem is this. When I take my tube out, I do see a nice white pellet of my nuclear DNA, but there is also some wispy gooey stuff at the surface of my supernatant. I've tried to re-spin, but this stuff never pellets. I don't want to continue my western with this stuff because I am sure it throws of my bradford measurements (to make sure I load equal amounts).

Any help would be appreciated. Thank you!

Wes

#2 Curtis

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Posted 09 August 2012 - 09:30 PM

This is a common problem and has been asked many times on this forum before. You need to add 1-2 ul DNase to your sample. Even sonication won't help you. But if you do all your work on ice you are less likely to see that gooey clump.

#3 Druss00

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Posted 09 August 2012 - 09:57 PM

This is a common problem and has been asked many times on this forum before. You need to add 1-2 ul DNase to your sample. Even sonication won't help you. But if you do all your work on ice you are less likely to see that gooey clump.


Cool, and the np40 doesn't hinder the DNAse? Also, to make myself clear I'm collectin supernatant and not the nuclear pellet. Thanks!

#4 Curtis

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Posted 09 August 2012 - 11:42 PM

I know

#5 bob1

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Posted 10 August 2012 - 03:17 PM

You add straight NP-40 to your cells? Not some sort of lysis buffer containing NP-40?

#6 Druss00

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Posted 10 August 2012 - 09:05 PM

No, Use the NP-40 lysis buffer (I forget the recipe)

#7 bob1

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Posted 10 August 2012 - 09:11 PM

Ah, that makes more sense. The more cells you are trying to lyse in the the volume, the more likely you are to get the DNA problem.. Typically 10^4 -10^5 cells per ul is OK, but anthing more than this tends to get the sticky DNA.

Some DNases are inhibited by detergents such as NP-40, I think benzonase will still work.

#8 Druss00

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Posted 10 August 2012 - 09:29 PM

Thanks bob1 and Curtis!





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