Anti-Integrin beta 1 antibody [12G10] (ab30394)

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.

ab30394 staining Integrin beta 1 in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-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 overnight at 4°C with ab30394 at 5µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

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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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.