Anti-MBD3 antibody [EPR9913] - ChIP Grade

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-MBD3 antibody [EPR9913] - ChIP Grade (AB157464)

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) labelling MBD3 with purified ab157464 at 1/1000. Cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (ab150077). Nuclei counterstained with DAPI (blue).

Control : PBS only

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-MBD3 antibody [EPR9913] - ChIP Grade (AB157464)

Immunofluorescent analysis of HeLa cells labeling MBD3 with ab157464 at 1/100 dilution.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MBD3 antibody [EPR9913] - ChIP Grade (AB157464)

Intracellular flow cytometric analysis of permeabilized 293T cells labeling MBD3 with ab157464 at 1/10 dilution (red) compared to a rabbit IgG negative control (green).

• ChIP

Unknown

ChIP - Anti-MBD3 antibody [EPR9913] - ChIP Grade (AB157464)

Chromatin was prepared from HeLa cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab157464 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.
*http : //www.abcam.com/resources?keywords=X%20ChIP%20protocol

• ChIP

Unknown

ChIP - Anti-MBD3 antibody [EPR9913] - ChIP Grade (AB157464)

Chromatin was prepared from NIH/3T3 cells according to the Abcam Dual X-ChIP protocol*. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab157464 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.
*http : //www.abcam.com/resources?keywords=X%20ChIP%20protocol

• WB

Lab

Western blot - Anti-MBD3 antibody [EPR9913] - ChIP Grade (AB157464)

Lanes 1 - 4 : Merged signal (red and green). Green - ab157464 observed at 33 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab157464 was shown to recognize MBD3 when MBD3 knockout samples were used, along with additional cross-reactive bands. Wild-type and MBD3 knockout samples were subjected to SDS-PAGE. ab157464 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-MBD3 antibody [EPR9913] - ChIP Grade (ab157464) at 1/1000 dilution

Lanes 1 and 3:

Wild-type HAP1 cell lysate at 20 µg

Lanes 2 and 4:

MBD3 knockout HAP1 cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed at 1/10000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed at 1/10000 dilution

Predicted band size: 33 kDa

Observed band size: 33 kDa

false

• WB

Unknown

Western blot - Anti-MBD3 antibody [EPR9913] - ChIP Grade (AB157464)

All lanes:

Western blot - Anti-MBD3 antibody [EPR9913] - ChIP Grade (ab157464) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

293T cell lysate at 10 µg

Lane 3:

Fetal brain lysate at 10 µg

Lane 4:

Y79 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 33 kDa

false