Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade (AB236639)

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) 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab236639).

• IP

Unknown

Immunoprecipitation - Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade (AB236639)

RUNX2 was immunoprecipitated from 0.35 mg PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate 10ug 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 (human prostate adenocarcinoma epithelial cell) 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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab236639).

All lanes:

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

Predicted band size: 57 kDa

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• IP

Unknown

Immunoprecipitation - Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade (AB236639)

RUNX2 was immunoprecipitated from 0.35 mg Saos-2 (human osteosarcoma epithelial) whole cell lysate 10ug 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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab236639).

All lanes:

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

Predicted band size: 57 kDa

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• ChIP

Lab

ChIP - Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade (AB236639)

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade (AB236639)

Immunohistochemical analysis of paraffin-embedded rat embryo 14.5 day tissue 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab236639).

• WB

Lab

Western blot - Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade (AB236639)

Western blot : Anti-RUNX2 antibody [EPR22858-106] (ab236639) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab236639 was shown to bind specifically to RUNX2. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

All lanes:

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

Lane 1:

Saos-2 cell lysate at 20 µg

Lane 2:

MC3T3-E1 undifferentiated cell lysate at 20 µg

Lane 3:

MC3T3-E1 7-day Osteogenic differentiation cell lysate, at 20 µg

Lane 4:

MC3T3-E1 14-day Osteogenic differentiation cell lysate at 20 µg

Lane 5:

MC3T3-E1 28-day Osteogenic differentiation cell lysate at 20 µg

Lane 6:

C2C12 cell lysate at 20 µg

Lane 7:

SH-SY5Y cell lysate at 20 µg

Lane 8:

NIH/3T3 cell lysate at 20 µg

Lane 9:

LNCaP cell lysate at 20 µg

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• WB

Lab

Western blot - Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade (AB236639)

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

All lanes:

Western blot - Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade (ab236639) at 1/1000 dilution

Lane 1:

PC-3 (human prostate adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

MDA-MB-231 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 3:

Saos-2 (human osteosarcoma epithelial), whole cell lysate at 20 µg

Lane 4:

MEF (mouse embryonic fibroblast (immortalized)), whole cell lysate at 20 µg

Lane 5:

C2C12 (mouse myoblasts myoblast), whole cell lysate at 20 µg

Lane 6:

MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 7:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 57 kDa

Observed band size: 60 kDa,64 kDa

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• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade (AB236639)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5X10^5 of positive cell line Saos-2 or low expression cell line HeLa were used along with 5µg of ab236639 [EPR22858-106]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Lab

Western blot - Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade (AB236639)

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

Exposure time : 3 mintues

All lanes:

Western blot - Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade (ab236639) at 1/1000 dilution

Lane 1:

C6 (rat glial tumor glial cell), whole cell lysate at 20 µg

Lane 2:

PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 57 kDa

Observed band size: 60 kDa,64 kDa

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• WB

CiteAb

Western blot - Anti-RUNX2 antibody [EPR22858-106] - ChIP Grade (AB236639)

RUNX2 western blot using anti-RUNX2 antibody [EPR22858-106] - ChIP Grade ab236639. Publication image and figure legend from Luo, Y., Xia, H., et al., 2022, Evid Based Complement Alternat Med, PubMed 35769158.

ab236639 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab236639 please see the product overview.

The changes in bone metabolism parameters and serological markers in rats after 12 weeks of JWYHD intragastric treatment are described. (a) Micro-CT was used to scan three-dimensional reconstruction images of bone trabeculae. (b) BMD. (c) BV/TV. (d) Tb.Th. (e) Tb.N. (f) Tb.Sp. (g) The translocation expression of BMP2, a key protein in the pathway, was detected by immunofluorescence. (h) The protein expression was measured by WB after culturing for 1 week. (i) BMP2 translocation percentage. (j) Weight gain of rats. (k) ELISA quantified the content of bone resorption marker β-CTX in serum. (l) ELISA quantified the content of the bone formation marker P1NP in serum. (m)-(q) The quantitative measurement of protein expression (BMP2, P-SMAD1/5/8, RUNX2, OCN, PPARγ). The data were represented by mean ± SD (n = 3), p < 0.05 were considered as significant difference : * VS. SHAM group, #VS. M group.

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