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How to pull down chromatin and identify protein associated with it?


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#1 Schweinsteiger

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Posted 15 December 2010 - 09:38 PM

Hi, everyone,

I want to identify proteins associated with the chromatin region near a specific endonuclease induced double-strand break.

I know ChIP was broadly used to pull down proteins and identify DNA associated with them. I want to know if there are some methods which can do the reverse work?

For example, can we use labeled DNA probe (complementary to the ssDNA or dsDNA near the DNA break) to pull down a fragment of chromatin (through complemantary match) and then identify the proteins crosslinked with that chomatin?

or can we label the ssDNA end of the break and then pull it down?

As the repair factors can not bind sythesized oligos, so I can not use sythesized and labeled oligos to pull down these proteins directly.

Is there any method to do this or can we develop some reasonable methods?

Many thanks!

Bastian

#2 Blondterror

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Posted 25 January 2011 - 08:45 AM

Hi, everyone,

I want to identify proteins associated with the chromatin region near a specific endonuclease induced double-strand break.

I know ChIP was broadly used to pull down proteins and identify DNA associated with them. I want to know if there are some methods which can do the reverse work?

For example, can we use labeled DNA probe (complementary to the ssDNA or dsDNA near the DNA break) to pull down a fragment of chromatin (through complemantary match) and then identify the proteins crosslinked with that chomatin?

or can we label the ssDNA end of the break and then pull it down?

As the repair factors can not bind sythesized oligos, so I can not use sythesized and labeled oligos to pull down these proteins directly.

Is there any method to do this or can we develop some reasonable methods?

Many thanks!

Bastian



Another approach you could try is to acoustically shear your sample instead of enyzmatically... then if you have an Ab to your protein you should be able to pull it down. Covaris has protocols to due this method using one of their AFA based instruments. You can control the shearing so your protein will be left intact.

Chris




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