Anti-CHD4 antibody [EPR22953-38] - ChIP Grade

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade (AB240640)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labeling CHD4 with ab240640 at 1/500 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing strong nuclear staining and weak cytoplasmic staining in HeLa cell line is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade (AB240640)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling CHD4 with ab240640 at 1/600 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) / Black isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 was used as the secondary antibody.

• ChIP

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ChIP - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade (AB240640)

Chromatin was prepared from K-562 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 ab240640 (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 from paper PMID : 23505388.

• IP

Unknown

Immunoprecipitation - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade (AB240640)

CHD4 was immunoprecipitated from 0.35 mg NCCIT (Human pluripotent embryonic carcinoma epithelial cell) whole cell lysate 10ug with ab240640 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab240640 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : NCCIT whole cell lysate 10ug.

Lane 2 : ab240640 IP in NCCIT whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab240640 in NCCIT whole cell lysate.

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

Exposure time : 3 min.

All lanes:

Immunoprecipitation - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade (ab240640)

Predicted band size: 218 kDa

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• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade (AB240640)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeling CHD4 with ab240640 at 1/500 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing strong nuclear staining and weak cytoplasmic staining in NIH/3T3 cell line is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

• WB

Lab

Western blot - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade (AB240640)

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : PMID : 28572115) .

Exposure time : 1 second.

All lanes:

Western blot - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade (ab240640) at 1/1000 dilution

Lane 1:

NCCIT (human pluripotent embryonic carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

HEK-293 (human embryonic kidney epithelial cell), whole cell lysate at 20 µg

Lane 5:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 218 kDa

Observed band size: 280 kDa

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• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade (AB240640)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 K562 (human chronic myelogenous leukemia lymphoblast) cells and 5 µg of ab240640 [EPR22953-38]. 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. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade (AB240640)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 K562 (human chronic myelogenous leukemia lymphoblast) cells and 5 µg of ab240640 [EPR22953-38]. 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. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade (AB240640)

ChIC/CUT&RUN was performed using a pAG-MNase at a final concentration of 700 ng/mL, 2.5 x 10^5 K562 (human chronic myelogenous leukemia lymphoblast) cells and 5 µg of ab240640 [EPR22953-38]. 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. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.