Lanes 1, 5 and 9: Wild-type HAP1 cell lysate (20 µg)
Lanes 2, 6 and 10: Integrin alpha 2 knockout HAP1 cell lysate (20 µg)
Lanes 3, 7 and 11: A431 cell lysate (20 µg)
Lanes 4, 8 and 12: T47D cell lysate (20 µg)
Lanes 1, 2, 3 and 4: Green signal from target - ab181548 observed at 150 kDa
Lanes 5, 6, 7 and 8: Red signal from loading control - ab8245 observed at 37 kDa
Lanes 9, 10, 11 and 12: Merged (red and green) signal

ab181548 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. ab181548 and ab8245 (loading control to GAPDH) were diluted 1/5000 and 1/2000 respectively and incubated overnight at 4°C. 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 1/10 000 dilution for 1 h at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded Human squamous cell carcinoma of cervix tissue labeling Integrin alpha 2 with ab181548 at 1/500 dilution followed by Goat Anti-Rabbit HRP (IgG H&L) (ab97051) at 1/500 dilution. Membrane and weak cytoplasmic staining on epithelial cells of human squamous cell carcinoma of cervix tissue is observed. Counter stained with Hematoxylin.
Negative control: Used PBS instead of primary antibody.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

ab181548 staining Integrin α2 in wild-type HAP1 cells (top panel) and Integrin α2 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab181548 at 1μg/ml concentration and ab7291 at 1ug/ml concentration overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to Mouse IgG (Alexa Fluor® 594) (ab150120) at 2ug/ml (shown in pseudo-color red). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

All lanes : Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (ab181548) at 1/20000 dilution

Lane 1 : A549 (Human lung carcinoma) whole cell lysates
Lane 2 : A431 (Human epidermoid carcinoma) whole cell lysates
Lane 3 : 293T (Human epithelial cells from embryonic kidney) whole cell lysates
Lane 4 : T-47D (Human ductal breast epithelial tumor cell line) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated 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 and diluting buffer 5% NFDM/TBST.

The increased molecular mass observed is due to glycosylation.

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Integrin alpha 2 with ab181548 at 1/500 dilution followed by Goat Anti-Rabbit HRP (IgG H&L) (ab97051) at 1/500 dilution. Membrane and weak cytoplasmic staining on epithelial cells of human colon is observed. Counter stained with Hematoxylin.

Negative control: Used PBS instead of primary antibody.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF7 (Human breast adenocarcinoma cell line) cells labeling integrin alpha 2 with ab181548 at 1/100 dilution, followed by Goat anti-rabbit IAlexa Fluor® 488 (IgG) (ab150077) secondary antibody at 1/400 dilution (green). Confocal image showing membrane staining on MCF7 cell line is observed. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (goat anti-mouse AlexaFluor®594 secondary antibody) at 1/500 dilution (red).
The negative controls are as follows:-
-ve control 1 - ab181548 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2. - ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

 

All lanes : Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (ab181548) at 1/5000 dilution

Lane 1 : Mouse heart tissue lysate
Lane 2 : Mouse kidney tissue lysate
Lane 3 : Rat spleen tissue lysate
Lane 4 : C6 (Rat glial tumor cells) whole cell lysate
Lane 5 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated 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 and diluting buffer 5% NFDM/TBST.
The increased molecular mass observed is due to glycosylation.

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling Integrin alpha 2 with ab181548 at 1/500 dilution followed by Goat Anti-Rabbit HRP (IgG H&L) (ab97051) at 1/500 dilution. Membrane and weak cytoplasmic staining on epithelial cells of Mouse kidney tubule is observed. Counter stained with Hematoxylin.
Negative control: Used PBS instead of primary antibody.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized PC-3 (Human prostate adenocarcinoma cell line) cells labeling integrin alpha 2 with ab181548 at 1/100 dilution, followed by Goat anti-rabbit IAlexa Fluor® 488 (IgG) (ab150077) secondary antibody at 1/400 dilution (green). Confocal image showing membrane and weakly cytoplasmic staining on PC-3 cell line is observed. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (goat anti-mouse AlexaFluor®594 secondary antibody) at 1/500 dilution (red).

The negative controls are as follows:-
-ve control 1 - ab181548 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2. - ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

 

Flow cytometric analysis of 2% paraformaldehyde-fixed A549 (Human lung carcinoma) cells labeling integrin alpha 2 with ab181549 at 1/160 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and a unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling Integrin alpha 2 with ab181548 at 1/500 dilution followed by Goat Anti-Rabbit HRP (IgG H&L) (ab97051) at 1/500 dilution. Membrane staining on epithelial cells of Rat colon tissue is observed. Counter stained with Hematoxylin.
Negative control: Used PBS instead of primary antibody.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (ab181548) at 1/5000 dilution

Lane 1 : Human fetal brain whole cell lysates
Lane 2 : Human fetal heart whole cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG 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 and diluting buffer 5% NFDM/TBST.
The increased molecular mass observed is due to glycosylation.

Integrin alpha 2 was immunoprecipitated from 1mg of T-47D (Human ductal breast epithelial tumor cell line) whole cell extract with ab181548 at 1/150 dilution. Western blot was performed using ab181548 at 1/20,000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution. Lane 1: T-47D whole cell extract  Lane 2: PBS instead of T-47D whole cell extract.

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