All lanes : Anti-Paxillin antibody [Y113] (ab32084) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PXN knockout HeLa cell lysate
Lane 3 : HEK-293 cell lysate
Lane 4 : A431 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 68 kDa
Observed band size: 68 kDa

Lanes 1-4: Merged signal (red and green). Green - ab32084 observed at 68 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab32084 Anti-Paxillin antibody [Y113] was shown to specifically react with Paxillin in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264767 (knockout cell lysate ab257044) was used. Wild-type and Paxillin knockout samples were subjected to SDS-PAGE. ab32084 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) were incubated overnight at 4°C at 1 in 1000 dilution and 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.

 

Image: Courtesy of Dr. Shaohua Li, UMDNJ-Robert Wood Johnson Medical School

Sample: mouse embryonic fibroblasts

Preparation:

Fix in 3% PFA in PBS for 30 min at RT

Primary antibody: Rabbit anti-paxillin Y113 (ab32084), 1:100

Secondary antibody: Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488) pre-adsorbed (ab150081), 1:200

Rhodamine-phalloidin, 1:100

Nuclei were counterstained with DAPI

 

Flow cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling with purified ab32084 at 1/100 dilution ( 10ug/ml) (Red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488)(ab150077) )(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black)(ab172730) was used as a isotype control.Cell without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

Equilibrium disassociation constant (K_D)
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Anti-Paxillin antibody [Y113] (ab32084) at 1/10000 dilution + HeLa cell lysate.

Predicted band size: 68 kDa
Observed band size: 60 kDa why is the actual band size different from the predicted?

Shear-induced Cell Remodeling.

3T3 fibroblasts are shown under the indicated cation and shear conditions. The shear direction in each image is indicated by a white arrow. Images show paxillin in green (ab32084), the actin cytoskeleton in red, and the nucleus (DNA) in blue. The approximate pre-shear cell area is indicated by white dashed lines as determined from the focal adhesions that remained on the substrate, which are indicated by open arrowheads. The bottom left image was contrast-enhanced 2-fold to better visualize the focal adhesions that remained on the substrate. Inset images are shown from regions outlined in white.

ab32084 staining paxillin in MEF1 cells treated with (+/-)-blebbistatin (ab120425), by ICC/IF. Decreased membrane expression of paxillin correlates with increased concentration of (+/-)-blebbistatin, as described in literature.
The cells were incubated at 37°C for 1h in media containing different concentrations of ab120425 ((+/-)-blebbistatin) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab32084 (1/100 dilution) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab32084 staining paxillin in MEF1 cells treated with (S)-(-)-Blebbistatin (ab120491), by ICC/IF. Decreased membrane expression of paxillin correlates with increased concentration of (S)-(-)-Blebbistatin , as described in literature.
The cells were incubated at 37°C for 2h in media containing different concentrations of ab120491 ( (S)-(-)-Blebbistatin ) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab32084 (1/100 dilution) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.