All lanes : Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade (ab236639) at 1/1000 dilution

Lane 1 : PC-3 (human prostate adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : MDA-MB-231 (human breast adenocarcinoma epithelial cell), whole cell lysate
Lane 3 : Saos-2 (human osteosarcoma epithelial), whole cell lysate
Lane 4 : MEF (mouse embryonic fibroblast (immortalized)), whole cell lysate
Lane 5 : C2C12 (mouse myoblasts myoblast), whole cell lysate
Lane 6 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate
Lane 7 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 57 kDa
Observed band size: 60,64 kDa why is the actual band size different from the predicted?

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

 

All lanes : Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade (ab236639) at 1/1000 dilution

Lane 1 : C6 (rat glial tumor glial cell), whole cell lysate
Lane 2 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 57 kDa
Observed band size: 60,64 kDa why is the actual band size different from the predicted?

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