Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Integrin alpha 2 knockout HAP1 cell lysate (20 µg)
Lane 3: A431 cell lysate (20 µg)
Lane 4: T47D cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab133557 observed at 165 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab133557 was shown to specifically react with Integrin alpha 2 when Integrin alpha 2 knockout samples were used. Wild-type and Integrin alpha 2 knockout samples were subjected to SDS-PAGE. ab133557 and ab8245 (loading control to GAPDH) were diluted 1/10 000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed withGoat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 h at room temperature before imaging.

All lanes : Anti-Integrin alpha 2 antibody [EPR5788] (ab133557) at 1/10000 dilution (purified)

Lane 1 : T47-D cell lysate
Lane 2 : HEK293 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 129 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Anti-Integrin alpha 2 antibody [EPR5788] (ab133557) at 1/50000 dilution (purified) + A431 cell lysate at 20 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 129 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Anti-Integrin alpha 2 antibody [EPR5788] (ab133557) at 1/10000 dilution (purified) + Mouse spleen tissue lysate at 20 µg

Secondary
Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 129 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

All lanes : Anti-Integrin alpha 2 antibody [EPR5788] (ab133557) at 1/10000 dilution (unpurified)

Lane 1 : T47D lysate
Lane 2 : 293T lysate
Lane 3 : Human platelet lysate
Lane 4 : A431 lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 129 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling Integrin alpha 2 with purified ab133557 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue labelling Integrin alpha 2 with unpurified ab133557 at 1/250.

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

Flow Cytometry analysis of A549 cells labelling Integrin alpha 2 with purified ab133557 at 1/60 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

Overlay histogram showing A431 cells stained with unpurified ab133557 (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 (ab133557, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-rabbit Alexa Fluor^® 488 (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.

Equilibrium disassociation constant (K_D)
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