All lanes : Anti-Integrin alpha 2 antibody [EPR17349] (ab181549) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ITGA2 knockout HeLa cell lysate
Lane 3 : A431 cell lysate
Lane 4 : K562 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 129 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab181549 observed at 160 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab181549 Anti-Integrin alpha 2 antibody [EPR17349] was shown to specifically react with Integrin alpha 2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264819 (knockout cell lysate ab257262) was used. Wild-type and Integrin alpha 2 knockout samples were subjected to SDS-PAGE. ab181549 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 Dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-Integrin alpha 2 antibody [EPR17349] (ab181549) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : ITGA2 knockout HAP1 whole cell lysate
Lane 3 : A431 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 129 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab181549 observed at 160 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab181549 was shown to specifically recognize ITGA2 in wild-type HAP1 cells in addition to additional cross-reactive bands. No band was observed when ITGA2 knockout samples were examined. Wild-type and ITGA2 knockout samples were subjected to SDS-PAGE. Ab181549 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Integrin alpha 2 antibody [EPR17349] (ab181549) at 1/1000 dilution

Lane 1 : A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : A431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : PC-3 (Human prostate adenocarcinoma cell line) whole cell lysate
Lane 4 : MCF7 (Human breast adenocarcinoma cell line) 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: 129 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The increased of molecular weight is due to glycosylation and is consistent with what has been described in the literature (PMID: 10825287 & 22354032).

Anti-Integrin alpha 2 antibody [EPR17349] (ab181549) at 1/1000 dilution + Human kidney lysate at 20 µg

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

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


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The increased of molecular weight is due to glycosylation and is consistent with what has been described in the literature (PMID: 10825287 & 22354032).

Anti-Integrin alpha 2 antibody [EPR17349] (ab181549) at 1/5000 dilution + Human skin lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

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


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The increased of molecular weight is due to glycosylation and is consistent with what has been described in the literature (PMID: 10825287 & 22354032).

Integrin alpha 2 was immunoprecipitated from 0.35 mg of A549 (Human lung carcinoma cell line) whole cell lysate with ab181549 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab181549 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: A549 whole cell lysate, 10µg (Input).

Lane 2: ab181549 IP in A549 whole cell lysate.

Lane 3: Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab181549 in A549 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds.