Recombinant Anti-Integrin alpha 5 antibody [EPR7854] (ab150361)

Observed band size : 19, 150 kDa

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

Blocking/Diluting buffer 5% NFDM/TBST 

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.

Intracellular Flow Cytometry analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Integrin alpha 5 with purified ab150361 at 1/20 dilution (10µg/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). 

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

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

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

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

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 unpurifiedab150361 (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. 

Observed band size : 19, 150 kDa

Exposure time : 3 minutes

Blocking/Diluting buffer 5% NFDM/TBST