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?

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.

 

All lanes : Anti-Paxillin antibody [E228] (ab32115) at 1/10000 dilution

Lane 1 : Wild-type A431 whole cell lysate
Lane 2 : PXN knockout A431 whole cell lysate
Lane 3 : U-87 MG whole cell lysate
Lane 4 : PC-3 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 64 kDa
Observed band size: 65 kDa why is the actual band size different from the predicted?

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

ab32115 was shown to specifically react with PXN in wild-type A431 cells as signal was lost in PXN knockout cells. Wild-type and PXN knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab32115 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-Paxillin antibody [E228] (ab32115) at 1/1000 dilution (purified)

Lane 1 : HEK-293 (Human embryonic kidney) whole cell lysates
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
Lane 3 : C2C12 (Mouse myoblasts myoblast) whole cell lysates
Lane 4 : Rat-1 (Rat embryonic fibroblast) whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051)

Predicted band size: 64 kDa
Observed band size: 60,64 kDa why is the actual band size different from the predicted?

Based on the immunogen sequence blast, this antibody recognizes alpha, beta and gamma isoforms. The molecular weight observed is consistent with what has been described in the literature PMID: 9712867 and 20388733

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.

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.

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.

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).

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.

All lanes : Anti-Paxillin antibody [E228] (ab32115) at 1/1000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : HEK-293 (human embryonic kidney) whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 64 kDa
Observed band size: 64 kDa


Exposure time: 3 minutes

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

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.

All lanes : Anti-Paxillin antibody [E228] (ab32115) at 1/10000 dilution

Lane 1 : untreated NIH 3T3 cell lysate
Lane 2 : PDGF treated NIH 3T3 cell lysate

Predicted band size: 64 kDa
Observed band size: 64 kDa

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.

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.

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