Anti-Integrin alpha 2 抗体 [EPR17338] - C-terminal

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (AB181548)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (AB181548)

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).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (AB181548)

Flow cytometry overlay histogram showing staining with ab181548 of HAP1 WT positive cells (magenta line) and HAP1-ITGA2 KO negative cells (red line). The cells were fixed with 80% methanol, and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab181548) (1x 10⁶ in 100μl at 0.2μg/ml (1/2,500)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C.

Isotype control antibody was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control in HAP1 WT cells (black line) and HAP1-ITGA2 KO cells (grey line), used at the same concentration and conditions as the primary antibody.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (AB181548)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (AB181548)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (AB181548)

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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (AB181548)

Intracellular 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.

• IP

Supplier Data

Immunoprecipitation - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (AB181548)

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.

All lanes:

Immunoprecipitation - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (ab181548)

Predicted band size: 129 kDa

Observed band size: 150 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (AB181548)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (AB181548)

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.

• WB

Supplier Data

Western blot - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (AB181548)

Blocking and diluting buffer 5% NFDM/TBST.
The increased molecular mass observed is due to glycosylation.

All lanes:

Western blot - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (ab181548) at 1/5000 dilution

Lane 1:

Human fetal brain whole cell lysates at 10 µg

Lane 2:

Human fetal heart whole cell lysates at 10 µg

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

false

• WB

Supplier Data

Western blot - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (AB181548)

Blocking and diluting buffer 5% NFDM/TBST.

The increased molecular mass observed is due to glycosylation.

All lanes:

Western blot - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (ab181548) at 1/20000 dilution

Lane 1:

A549 (Human lung carcinoma) whole cell lysates at 20 µg

Lane 2:

A431 (Human epidermoid carcinoma) whole cell lysates at 20 µg

Lane 3:

293T (Human epithelial cells from embryonic kidney) whole cell lysates at 20 µg

Lane 4:

T-47D (Human ductal breast epithelial tumor cell line) whole cell lysates at 20 µg

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

false

• WB

Unknown

Western blot - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (AB181548)

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.

All lanes:

Western blot - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (ab181548)

Predicted band size: 129 kDa

false

• WB

Supplier Data

Western blot - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (AB181548)

Blocking and diluting buffer 5% NFDM/TBST.
The increased molecular mass observed is due to glycosylation.

All lanes:

Western blot - Anti-Integrin alpha 2 antibody [EPR17338] - C-terminal (ab181548) at 1/5000 dilution

Lane 1:

Mouse heart tissue lysate at 10 µg

Lane 2:

Mouse kidney tissue lysate at 10 µg

Lane 3:

Rat spleen tissue lysate at 10 µg

Lane 4:

C6 (Rat glial tumor cells) whole cell lysate at 10 µg

Lane 5:

NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate at 10 µg

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

false