how to validated CHIP after H3k27 3Me - (Mar/13/2007 )
how to validated CHIP after H3k27 3Me antibody pull down?
we did chip in primary tumor sample, but we do not know which gene can work as a positive contrl for H3K27-me3, so we do not know if our chip works. because it is primary sample, we can not get information from literature. and the only way to find the candidate positive gene is to do chip itself, it becomes a paradox.
-gaodaxia-
There are some references for genes enriched for h3k27me3 in certain cell lines. However, in primary samples there maybe really no good control. You could try a gene that marked in ES cells and seem stable in later stages of differentiation
-tap14-
QUOTE (gaodaxia @ Mar 13 2007, 01:59 AM)
how to validated CHIP after H3k27 3Me antibody pull down?
we did chip in primary tumor sample, but we do not know which gene can work as a positive contrl for H3K27-me3, so we do not know if our chip works. because it is primary sample, we can not get information from literature. and the only way to find the candidate positive gene is to do chip itself, it becomes a paradox.
we did chip in primary tumor sample, but we do not know which gene can work as a positive contrl for H3K27-me3, so we do not know if our chip works. because it is primary sample, we can not get information from literature. and the only way to find the candidate positive gene is to do chip itself, it becomes a paradox.
A good bet is to choose a gene which is developmentally very restricted to a cell type other than the one you are working with (e.g. b-globin for non-hematopoietic cells or nanog for non-ES cells). Also, look for PREs (polycomb response elements) for instance in homeobox genes.
-KPDE-
QUOTE (KPDE @ Mar 13 2007, 06:26 PM)
QUOTE (gaodaxia @ Mar 13 2007, 01:59 AM)
how to validated CHIP after H3k27 3Me antibody pull down?
we did chip in primary tumor sample, but we do not know which gene can work as a positive contrl for H3K27-me3, so we do not know if our chip works. because it is primary sample, we can not get information from literature. and the only way to find the candidate positive gene is to do chip itself, it becomes a paradox.
we did chip in primary tumor sample, but we do not know which gene can work as a positive contrl for H3K27-me3, so we do not know if our chip works. because it is primary sample, we can not get information from literature. and the only way to find the candidate positive gene is to do chip itself, it becomes a paradox.
A good bet is to choose a gene which is developmentally very restricted to a cell type other than the one you are working with (e.g. b-globin for non-hematopoietic cells or nanog for non-ES cells). Also, look for PREs (polycomb response elements) for instance in homeobox genes.
Sorry to bring back an old topic....
I am also having trouble finding a good positive control for my H3K27Me3 ChIP
I am using primary white blood cells from patients...a rep from millipore told me that I could use MYO-D (muscle specific) as my positive control, but that shows no enrichment over input via real time PCR. I then found one paper that used Keratin17 as their positive control. I ordered the primers and gave it a go , and again nothing! The antibody I am using has been tested in another lab here, and all my H3K9Ac ChIPs are working! Hmmm...can anyone give me some advice?? Thanks in advance! 
-Clare-
QUOTE (Clare @ Feb 13 2008, 03:14 AM)
QUOTE (KPDE @ Mar 13 2007, 06:26 PM)
QUOTE (gaodaxia @ Mar 13 2007, 01:59 AM)
how to validated CHIP after H3k27 3Me antibody pull down?
we did chip in primary tumor sample, but we do not know which gene can work as a positive contrl for H3K27-me3, so we do not know if our chip works. because it is primary sample, we can not get information from literature. and the only way to find the candidate positive gene is to do chip itself, it becomes a paradox.
we did chip in primary tumor sample, but we do not know which gene can work as a positive contrl for H3K27-me3, so we do not know if our chip works. because it is primary sample, we can not get information from literature. and the only way to find the candidate positive gene is to do chip itself, it becomes a paradox.
A good bet is to choose a gene which is developmentally very restricted to a cell type other than the one you are working with (e.g. b-globin for non-hematopoietic cells or nanog for non-ES cells). Also, look for PREs (polycomb response elements) for instance in homeobox genes.
Sorry to bring back an old topic....
I am also having trouble finding a good positive control for my H3K27Me3 ChIP
I am using primary white blood cells from patients...a rep from millipore told me that I could use MYO-D (muscle specific) as my positive control, but that shows no enrichment over input via real time PCR. I then found one paper that used Keratin17 as their positive control. I ordered the primers and gave it a go , and again nothing! The antibody I am using has been tested in another lab here, and all my H3K9Ac ChIPs are working! Hmmm...can anyone give me some advice?? Thanks in advance! check out abcams webpage for their H3K27me3 ChIP antibody - even though the chip was done on hela cells, their controls can be useful
http://www.abcam.com/index.html?datasheet=6002#ab6002_9.jpg
i think its important to have a positive control and negative control gene in your sample, so instead of looking for enrichment above input, test for enrichment of positive genes above negative genes
although its always hard with primary samples
good luck!
-frozenlyse-
Keji Zhao recently published a whole genome analyses of histone marks in T cells.
Usually there are good positive controls for any experiment in the published literature.
High-Resolution Profiling of Histone Methylations in the Human Genome.
Cell, Volume 129, Issue 4, Pages 823-837
A. Barski, S. Cuddapah, K. Cui, T. Roh, D. Schones, Z. Wang, G. Wei, I. Chepelev, K. Zhao
-mikew-