All lanes : Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (ab128873) at 1/10000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma) whole cell lysate
Lane 2 : 293T (human embryonic kidney) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 83 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?



Blocking and diluting buffer 5% NFDM/TBST

Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (ab128873) at 1/10000 dilution + Mouse brain at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 83 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?



Blocking and diluting buffer 5% NFDM/TBST

Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (ab128873) at 1/10000 dilution + Rat heart at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 83 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?



Blocking and diluting buffer 5% NFDM/TBST

Chromatin was prepared from HeLa 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 ab128873 (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).

All lanes : Anti-CTCF antibody [EPR7314(B)] - ChIP Grade (ab128873) at 1/1000 dilution (un-purified)

Lane 1 : HeLa cell lysate
Lane 2 : 293T cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-Rabbit HRP at 1/2000 dilution

Predicted band size: 83 kDa

Immunohistochemical staining of paraffin-embedded human breast carcinoma sections labelling CTCF with purified ab128873 at dilution of 1:1000. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

Immunohistochemical staining of paraffin-embedded mouse kidney sections labelling CTCF with purified ab128873 at dilution of 1:1000. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

Immunohistochemical staining of paraffin-embedded rat kidney sections labelling CTCF with purified ab128873 at dilution of 1:1000. The secondary antibody used was ab97051; a goat anti-rabbit IgG H&L (HRP) at dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was performed using EDTA Buffer; pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

Immunohistochemical analysis of paraffin embedded Human colon tissue labelling CTCF with un-purified ab128873 at 1/250 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunocytochemistry/immunofluorescence staining of 4% paraformaldehyde fixed; 0.1% triton X 100 permeabilized HeLa (human cervix adenocarcinoma) cells with purified ab128873 at dilution of 1/500. The secondary antibody used was Alexa Fluor® 488; goat anti-rabbit IgG (ab150077) at a dilution of 1/1000. Nucleus was counter-stained with DAPI (blue). ab7291, a mouse anti-tubulin antibody (1/1000) was used to stain tubulin along with ab150120 (AlexaFluor®594 goat anti-mouse secondary, 1/1000) shown in the top right hand panel. The negative controls are shown in the bottom middle and right hand panels- for negative control 1 rabbit primary antibody and anti-mouse secondary antibody (ab150120) was used. For negative control 2 mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077) was used.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling CTCF with un-purified ab128873 at 1/250 dilution.

Flow cytometry analysis showing 4% paraformaldehyde fixed 293T (human embryonic kidney) cells labelling CTCF with purified ab128873 at dilution of 1/40 followed by the secondary antibody; Alexa Fluor® 488 goat-anti-rabbit IgG at dilution of 1/500 (red line). A non-specific IgG antibody (rabbit monoclonal) was used as isotype control (black line). The blue line shows cells without incubation with primary antibody and secondary antibody.

Overlay histogram showing Hela cells stained with un-purified ab128873 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab128873, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Equilibrium disassociation constant (K_D)
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