Anti-ATF2 antibody [E243] - ChIP Grade (ab32160) at 1/10000 dilution + HeLa cell lysate

Predicted band size: 54 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

Ab32160, at a dilution of 1/250, staining ATF2 in paraffin embedded breast carcinoma tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunocytochemistry analysis of A549 (human lung carcinoma epithelial cell) labeling ATF2 with purified ab32160 at 1/100 dilution (10 µg/ml). Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/1000 (2 µg/ml) was used as the secondary antibody. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.10 µg/ml) was used as counterstain. Nuclei were stained blue with DAPI.
Negative control: PBS instead of the primary antibody.

Chromatin was prepared from Jurkat (TPA and Ionomycin treated or not) cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab32160 (red), and 20µl of protein A/G sepharose beads slurry (10µl of sepharose A beads + 10µl of sepharose G beads). 5μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

ab32160 (purified) at 1/60 dilution (20 µg/mL) immunoprecipitating ATF2 in HeLa whole cell lysate.
Lane 3 (-): HeLa(Human cervix adenocarcinoma epithelial cell) whole cell lysate 10µgab32160 & HeLa whole cell lysateRabbit monoclonal IgG (ab172730) instead of ab32160 in HeLa whole cell lysate
For western blotting, ab32160 at 1/1000 dilution (2.284 µg/mL) and
bbit TureBlot: Anti-Rabbit IgG HRP was used as the secondary antibody at 1/1500 dilution.
Blocking and diluting buffer: 5% NFDM /TBST .

Flow cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling ATF2 (red) with ab32160 at a 1/2000 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.