All lanes : Anti-CHD4 antibody [EPR22953-38] - ChIP Grade (ab240640) at 1/1000 dilution

Lane 1 : NCCIT (human pluripotent embryonic carcinoma epithelial cell), whole cell lysate
Lane 2 : K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : HEK-293 (human embryonic kidney epithelial cell), whole cell lysate
Lane 5 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), 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: 218 kDa
Observed band size: 280 kDa why is the actual band size different from the predicted?

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.

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

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.

 

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.

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.

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.