Anti-KAT4 / TBP Associated Factor 1 antibody - ChIP Grade (ab28450)

BATCH NUMBER 253830 ORDER NUMBER 209721

DESCRIPTION OF THE PROBLEM No detectable enrichment at GAPDH or c-myc promoters in ChIP

SAMPLE human PC3 cell line (prostate epithelial)

PRIMARY ANTIBODY none

DETECTION METHOD qPCR is performed by 32P-dCTP incorporation.

POSITIVE AND NEGATIVE CONTROLS USED Negative control is a ChIP performed without antibody to control for background DNA recovery.

ANTIBODY STORAGE CONDITIONS I used this antibody the day it arrived right out of the box, remaining antibody was aliquoted and stored at -80C.

SAMPLE PREPARATION ChIP was performed on fragmented chromatin from ~10^7 cells in 1ml 20mM Tris pH 8.0, 150mM NaCl, 2mM EDTA, 1% Triton X-100 + 1X Complete Protease Inhibitor (Roche).

CROSSLINKING Cells were cross linked with 1% formaldehyde for 5min at room temp.

DNA FRAGMENTATION Chromatin was fragmented by 10 rounds of sonication in 30sec bursts.

IP STEP 5ug of antibody was added to chromatin, tubes were rotated 3hr at 4C, Protein A Sepharose beads were added to capture immunocomplexes and tubes were rotated overnight at 4C. Beads were washed sequentially with 3 wash buffers followed by 2 washes in TE (all containing protease inhibitors)

DECROSSLINKING Proteinase K (20ug, RNA-grade) digestion is performed in TE + 0.5% SDS at 55C for at least 1hr.

NaCl is added to 250mM, tubes are placed at 65C 4hr-overnight to reverse crosslinking.

DNA PURIFICATION DNA is purified by Phenol/Chloroform/Isoamyl Alcohol extraction followed by Ethanol precipitation.

PELLET AFTER PRECIPITATION DNA is precipitated in the presence of GlycoBlue, and pellets are clearly visible.

PRIMERS TESTED ON GENOMIC/INPUT DNA Primers are used to amplify both Input DNA and ChIP DNA in the PCR run.

NO-TEMPLATE CONTROL IN PCR no

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. This antibody has been applied by Abcams chromatin postdoc scientist by adding 5-10µg of antibody to 25µg of chromatin. Please can you tell me the mass of chromatin that you have been using in relation to the mas of chromatin; is this determined?

I was interested in the duration of fixation that you have been using. in my opinion a 5 minute duration is a little short, a duration that is suitable for the chromatin immunoprecipitation of histone modifications but not chromatin associated (transcription) factors. We commonly use a 10 minute duration of formaldehyde fixation. Our XChIP lab protocol can be located at the following link;

www.abcam.com/chip

I would also like to know the region of GAPDH that you are amplifying; the primers that my colleague uses amplifies the core promoter region of the GAPDH gene.

I look forward to hearing from you.