711418

antibody from Invitrogen Antibodies

Targeting: PXN

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

711418

Provider

Invitrogen Antibodies

Product name

Phospho-Paxillin (Ser178) Recombinant Polyclonal Antibody (21HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Monkey, Cat, Rat and Dog

Antibody clone number

21HCLC

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (30 µg lysate) of MDA-MB-231 (Lane 1), PC-3 (Lane 2), U-87 MG (Lane 3) and tissue extracts of Rat Brain (Lane 4), Mouse Lung (Lane 5). The blots were probed with Anti-Paxillin p (S178) Recombinant Rabbit Polyclonal Antibody (Product # 711418, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). Two isoforms of 61 and 64 kDa bands corresponding to Paxillin p (S178) was observed. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, MDA-MB-231 cells were fixed and permeabilized for detection of endogenous Paxillin pS178 using Anti- Paxillin pS178 Recombinant Rabbit Polyclonal Antibody (Product # 711418, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of Paxillin pS178 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear and cytoplasmic localization of Paxillin pS178. Panel e) shows loss of signal by competition with the Paxillin pS178 peptide, demonstrating antibody specificity. Panel f) demonstrates no competition with the non-phosphorylated peptide. Panel g) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.