All lanes : Anti-Integrin alpha 5 antibody [EPR7854] (ab150361) at 1/2000 dilution (Unpurified)

Lane 1 : Human bladder tissue lysate
Lane 2 : HT-1080 (Human urinary bladder carcinoma cell line) whole cell lysates
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysates
Lane 4 : U937 (Human histiocytic lymphoma cell line) whole cell lysates

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: 115 kDa

Observed band size : 19, 150 kDa

Exposure time : Lane 1-2: 1 minutes, Lane 3-4: 3 minutes

Blocking/Diluting buffer 5% NFDM/TBST 

Immunocytochemistry/ Immunofluorescence analysis of U937 (Human histiocytic lymphoma monocyte) cells labeling Integrin alpha 5 with purified ab150361 at 1:50 dilution (4.3 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemical analysis of paraffin embedded Human kidney tissue labelling Integrin alpha 5 with unpurified ab150361 antibody at a dilution of 1/100.

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

ab150361 (Purified) at 1:20 dilution (1 µg) immunoprecipitating Integrin alpha 5 in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate 10 µg
Lane 2 (+): ab150361 & HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab150361 in HeLa whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1:5000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Flow Cytometry analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Integrin alpha 5 with purified ab150361 at 1:20 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

All lanes : Anti-Integrin alpha 5 antibody [EPR7854] (ab150361) at 1/1000 dilution (Purified)

Lane 1 : NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysates
Lane 2 : MEF (Mouse embryonic fibroblast cell line) whole cell lysates
Lane 3 : C2C12 (Mouse myoblast cell line) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 115 kDa
Observed band size: 150,19 kDa why is the actual band size different from the predicted?

The bands detected by ab150361 are consistent with what have been described in PMID: 22740495. Full length: 150kDa; Light chain: 19kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cerebral cortex tissue sections labeling Integrin alpha 5 with purified ab150361 at 1:400 dilution (0.54 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-Integrin alpha 5 antibody [EPR7854] (ab150361) at 1/1000 dilution (Purified)

Lane 1 : Rat1 (Rat fibroblast cell line) whole cell lysates
Lane 2 : Rat liver lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 115 kDa
Observed band size: 150,19 kDa why is the actual band size different from the predicted?

The bands detected by ab150361 are consistent with what have been described in PMID: 22740495. Full length: 150kDa; Light chain: 19kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebral cortex tissue sections labeling Integrin alpha 5 with purified ab150361 at 1:400 dilution (0.54 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Anti-Integrin alpha 5 antibody [EPR7854] (ab150361) at 1/10000 dilution (Purified) + HT-1080 (Human fibrosarcoma cell line) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 115 kDa
Observed band size: 150,19 kDa why is the actual band size different from the predicted?

The bands detected by ab150361 are consistent with what have been described in PMID: 22740495. Full length: 150kDa; Light chain: 19kDa

Anti-Integrin alpha 5 antibody [EPR7854] (ab150361) at 1/1000 dilution (Purified) + U937 (Human histiocytic lymphoma cell line) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 115 kDa
Observed band size: 150,19 kDa why is the actual band size different from the predicted?

The bands detected by ab150361 are consistent with what have been described in PMID: 22740495. Full length: 150kDa; Light chain: 19kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebral cortex tissue sections labeling Integrin alpha 5 with purified ab150361 at 1:400 dilution (0.54 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

ab150361 staining Integrin alpha 5 in MCF7 (Human breast adenocarcinoma cell line) cells. The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Tween 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 unpurified ab150361 at 1μg/ml concentration and ab7291 at 1μg/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 2 μg/ml (showed in pseudocolour red) . Nuclear DNA was labelled in blue with DAPI.

unpurified ab150361 staining Integrin alpha 5 in wild-type HAP1 cells (top panel) and ITGA5 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Tween 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 ab150361 at 1/250 dilution and ab7291 at 1μg/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 2 μg/ml (showed in pseudocolour red) . Nuclear DNA was labelled in blue with DAPI.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U937 (Human histiocytic lymphoma cell line) cells labeling Integrin alpha 5 with unpurified ab150361 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor 488) (ab150077) secondary antibody at 1/400 dilution (green). Confocal image showing mainly membrane staining on U937 cell line. The nuclear counterstain is DAPI (blue).

Overlay histogram showing HeLa cells stained with unpurified ab150361 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab150361, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was 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. This antibody gave a positive signal in HeLa cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

All lanes : Anti-Integrin alpha 5 antibody [EPR7854] (ab150361) at 1/2000 dilution (Unpurified)

Lane 1 : Mouse heart tissue lysate
Lane 2 : Rat heart tissue 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: 115 kDa


Exposure time: 3 minutes

Observed band size : 19, 150 kDa

Exposure time : 3 minutes

Blocking/Diluting buffer 5% NFDM/TBST