Recombinant Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade (ab236639)

CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ab285373, 10⁵ HeLa cells and 5µg of Ab236639 [EPR22858-106]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads.  The ChIP data was conducted on chromatin prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10⁷ HeLa cells and 4 µg of Ab236639 [EPR22858-106]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.

Additional screenshots of mapped reads can be downloaded here.

Lysates were made freshly and used in WB test immediately to minimize protein degradation.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID:19419310)

Negative control: MCF-7 and HeLa (PMID: 20591170)

Exposure time: 70 seconds

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 19419310, 20591170)

Exposure time: 3 mintues

RUNX2 was immunoprecipitated from 0.35 mg Saos-2 (human osteosarcoma epithelial) whole cell lysate with ab236639 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab236639 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Saos-2 (human osteosarcoma epithelial) whole cell lysate 10ug

Lane 2: ab236639 IP in Saos-2 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab236639 in Saos-2 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds

RUNX2 was immunoprecipitated from 0.35 mg PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate with ab236639 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab236639 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: PC-3 whole cell lysate 10ug

Lane 2: ab236639 IP in PC-3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab236639 in PC-3 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds

Immunohistochemical analysis of paraffin-embedded human osteosarcoma tissue labeling RUNX2 with ab236639 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human osteosarcoma (PMID: 21731849) is observed. The section was incubated with ab236639 for 15 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded rat embryo 14.5 day tissue labeling RUNX2 with ab236639 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on the cartilage cells in the rat embryo 14.5 day tissue. The section was incubated with ab236639 for 15 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Legend Chromatin was prepared from Saos-2 cells according to the Abcam Dual-X-ChIP protocol. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min. 
The ChIP was performed with 25 µg of chromatin, 5 µg of ab236639 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). 
Primers and probes are commercial primers from PMCID: PMC3281617