Anti-Integrin beta 1 antibody [12G10] (ab30394) at 5 µg/ml + HT 1080 (Human fibrosarcoma) Whole Cell Lysate at 20 µg

Secondary
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 88 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?


Exposure time: 20 minutes


Integrin beta 1 contains a number of potential glycosylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.

ab30394 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 ab30394 at 10μg/ml concentration and ab202272 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 Mouse IgG (Alexa Fluor^® 488) (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labeled in blue with DAPI.

Overlay histogram showing HAP1 wildtype (green line) and HAP1-ITGB1 knockout cells (red line) stained with ab30394.

Live HAP1 wildtype and HAP1-ITGB1 knockout cells were incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab30394, 1µg/0.5x10⁶ cells) for 30 min at 22°C. A mouse IgG1 isotype control antibody (ab170190) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-ITGB1 knockout - grey line). Unlabeled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

ab30394 at 1/500 staining Integrin beta 1 in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells by Immunocytochemistry/ Immunofluorescence.

Cells were fixed in paraformaldehyde. The secondary used was an Alexa Fluor^® 488 Goat anti mouse (H + L), used at a 1/500 dilution. Actin filaments (Phalloidin-TRITC) and DNA (DAPI, blue) are also shown

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ab30394 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed in 4% formaldehyde for 10 minutes at room temperature and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab30394 at 10µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was a Goat Anti-Mouse Alexa Fluor^® 488 (IgG H&L) preadsorbed (ab150117) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor^® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

Overlay histogram showing HeLa (Human epithelial cell line from cervix adenocarcinoma) cells stained with ab30394 (red line).

The cells were fixed with 80% methanol (5 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 (ab30394, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight^® 488 (IgG; H+L) ab96879 at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

ab30394 at 10 µg/ml staining Integrin beta 1 in HT1080 (Human fibrosarcoma cell line) cells by Immunocytochemistry/ Immunofluorescence.

Cells were fixed in paraformaldehyde. The secondary used was an Alexa Fluor^® 555 Donkey anti mouse (H + L), used at a 1/500 dilution.