All lanes : Anti-Paxillin antibody [E228] (ab32115) at 1/10000 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: 64 kDa
Observed band size: 65 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 (ab32115).

Lanes 1-4: Merged signal (red and green). Green - ab32115 observed at 65 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab32115 Anti-Paxillin antibody [E228] 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. ab32115 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) were incubated overnight at 4°C at 1 in 10000 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.

 

 

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Paxillin with Purified ab32115 at 1:100 dilution (1.2 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32115).

ab32115 (purified) at 1:20 dilution (0.5µg) immunoprecipitating Paxillin in HEK-293 whole cell lysate.
Lane 1 (input): HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab32115 & HEK-293 whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32115 in HEK-293 whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32115).

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Paxillin with Purified ab32115 at 1:20 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32115).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast carcinoma tissue sections labeling Paxillin with Purified ab32115 at 1:50 dilution (2.34 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used. Negative control:PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32115).

Direct ELISA antigen dose-response curve using ab32115 at 0-1000 ng/mL. Antigen (human Paxillin phospho Y31 peptide/ unmodified peptide) concentration of 1000 ng/mL. An alkaline phosphatase-conjugated goat anti-rabbit IgG (H+L) (1/2500) was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32115).

Dot blot analysis of Paxillin (pY31) peptide (Lane 1) and Paxillin non-phospho peptide (Lane 2) labelling Paxillin with ab32115 at a dilution of 1/1000. ab97051 (Peroxidase conjugated goat anti-rabbit IgG (H+L)) was used as the secondary antibody at a dilution of 1/100000.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32115).

Immunohistochemical analysis of Paxillin expression in paraffin-embedded human colon carcinoma using 1/100 ab32115.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32115).

Immunofluorescence analysis of HeLa cells, staining Paxillin with ab32115.

Cells on the right were treated with MGAT1 shRNA. Cells were fixed with 2% paraformaldehyde, permeabilized using 0.2% Triton-X-100 and blocked by 5% BSA for 1 hour. Cells were incubated with primary antibody (1/400) overnight at 4°C. A FITC-conjugated donkey anti-rabbit IgG (1/500) was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32115).

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Paxillin with ab32115 at 1/100 (1 μg/ml). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000, 2 μg/ml) was used as the secondary antibody. The cells were counterstained with ab195889, anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1/200, 2.5 μg/ml. Nuclei counterstained with DAPI (blue). 

Confocal image showing membranous staining on HeLa cells.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32115).