All lanes : Anti-Integrin beta 1 antibody [EPR1040Y] (ab134179) at 1/500 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ITGB1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 88 kDa
Observed band size: 140-150 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab134179).

  Lanes 1- 2: Merged signal (red and green). Green - ab134179 observed at 140-150 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab134179 was shown to react with ITGB1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255354 (knockout cell lysate ab263847) was used. Wild-type HeLa and ITGB1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab134179 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 500 Dilution and a 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.

ab134179 staining Integrin beta 1 in wild-type HAP1 cells (top panel) and Integrin beta 1 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), 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 ab134179 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) 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). Nuclear DNA was labelled in blue with DAPI.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134179).

Overlay histogram showing MCF7 cells stained with ab134179 (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 (ab134179, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was a 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 data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134179).

Lanes 1-4 : Anti-Integrin beta 1 antibody [EPR1040Y] (ab134179) at 1/500 dilution
Lanes 5-8 : Anti-GAPDH antibody [6C5] - Loading Control (ab8245) at 1/2000 dilution

Lanes 1 & 5 : Wild-type HAP1 cell lysate
Lanes 2 & 6 : Integrin beta 1 knockout HAP1 cell lysate
Lanes 3 & 7 : U87-MG cell lysate
Lanes 4 & 8 : A431 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 88 kDa

This data was developed using the same antibody clone in a different buffer formulation (ab134179).

Lanes 1, 2, 3 and 4: Green signal from target – ab134179 observed at 140kDa
Lanes 5, 6, 7 and 8: Red signal from loading control – ab8245 observed at 37kDa
Lanes 9, 10, 11 and 12: Merged (red and green) signal

ab134179 was shown to specifically react with Integrin beta 1 in wild-type HAP1 cells. No band was observed when Integrin beta 1 knockout samples were examined. Wild-type and Integrin beta 1 knockout samples were subjected to SDS-PAGE. ab134179 and ab8245 (loading control to GAPDH) were diluted 1/500 and 1/2000 respectively and incubated overnight at 4ºC. Blots were developed withGoat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1hr at room temperature before imaging.

Immunohistochemical analysis of paraffin embedded Human hepatocellular carcinoma tissue labelling Integrin beta 1 with ab134179 antibody at a dilution of 1/100. HIER was performed with citrate buffer (pH 6.0).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134179).

Immunohistochemical analysis of paraffin embedded Human brain (endothelial cells) tissue labelling Integrin beta 1 with ab134179 antibody at a dilution of 1/100.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab134179).