Anti-Integrin alpha 2 antibody [EPR5788]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Integrin alpha 2 antibody [EPR5788] (AB133557)

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 Fluorr^® 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Integrin alpha 2 antibody [EPR5788] (AB133557)

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 Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Integrin alpha 2 antibody [EPR5788] (AB133557)

Intracellular 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Integrin alpha 2 antibody [EPR5788] (AB133557)

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.

• WB

Lab

Western blot - Anti-Integrin alpha 2 antibody [EPR5788] (AB133557)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Integrin alpha 2 antibody [EPR5788] (ab133557) at 1/50000 dilution

All lanes:

A431 cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 129 kDa,162 kDa

Observed band size: 150 kDa,162 kDa

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• WB

Lab

Western blot - Anti-Integrin alpha 2 antibody [EPR5788] (AB133557)

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:

Western blot - Anti-Integrin alpha 2 antibody [EPR5788] (ab133557)

Predicted band size: 129 kDa

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• WB

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Western blot - Anti-Integrin alpha 2 antibody [EPR5788] (AB133557)

All lanes:

Western blot - Anti-Integrin alpha 2 antibody [EPR5788] (ab133557) at 1/10000 dilution

Lane 1:

T47D lysate at 10 µg

Lane 2:

293T lysate at 10 µg

Lane 3:

Human platelet lysate at 10 µg

Lane 4:

A431 lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 129 kDa

Observed band size: 150 kDa

false

• WB

Lab

Western blot - Anti-Integrin alpha 2 antibody [EPR5788] (AB133557)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Integrin alpha 2 antibody [EPR5788] (ab133557) at 1/10000 dilution

Lane 1:

T47-D cell lysate at 20 µg

Lane 2:

HEK293 cell lysate at 20 µg

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

false

• WB

Lab

Western blot - Anti-Integrin alpha 2 antibody [EPR5788] (AB133557)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-Integrin alpha 2 antibody [EPR5788] (ab133557) at 1/10000 dilution

All lanes:

Mouse spleen tissue lysate at 20 µg

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

false

• OI-RD Scanning

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OI-RD Scanning - Anti-Integrin alpha 2 antibody [EPR5788] (AB133557)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.