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?

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 Integrin beta 1 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.

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.

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.

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

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.

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

Lane 1 : U937 cell lysate
Lane 2 : HeLa cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 88 kDa

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.

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.