Anti-CHD4 antibody [EPR22953-38] - ChIP Grade - BSA and Azide free

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade - BSA and Azide free (AB263025)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab240640).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade - BSA and Azide free (AB263025)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab240640).

• ChIP

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ChIP - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade - BSA and Azide free (AB263025)

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.
*http : //www.abcam.com/resources?keywords=X%20ChIP%20protocol This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab240640).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade - BSA and Azide free (AB263025)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labeing 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab240640).

• ChIC/CUT&RUN-seq

Lab

ChIC/CUT&RUN sequencing - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade - BSA and Azide free (AB263025)

This data was developed using the same antibody clone in a different buffer formulation (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 - BSA and Azide free (AB263025)

This data was developed using the same antibody clone in a different buffer formulation (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 - BSA and Azide free (AB263025)

This data was developed using the same antibody clone in a different buffer formulation (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.

• IP

Unknown

Immunoprecipitation - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade - BSA and Azide free (AB263025)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab240640).

All lanes:

Immunoprecipitation - Anti-CHD4 antibody [EPR22953-38] - ChIP Grade (<a href='/en-us/products/primary-antibodies/chd4-antibody-epr22953-38-chip-grade-ab240640'>ab240640</a>)

Predicted band size: 218 kDa

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