711694

antibody from Invitrogen Antibodies

Targeting: PAK5 KIAA1264, PAK7

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

711694

Provider

Invitrogen Antibodies

Product name

Phospho-PAK4/6/7 (Ser474) Recombinant Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

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 HeLa (Lane 1), A549 (Lane 2) and MCF-7 (Lane 3). The blots were probed either, with Anti-Phospho-PAK4 + PAK6 + PAK7 (pSer474) Recombinant Rabbit Polyclonal Antibody (Product # 711694, 1 µg/mL). To confirm the specificity competition was performed with the phosphopeptide (10 µg/mL) (Lane 4, 5, 6) and non phosphopeptide (10 µg/mL) (Lane 7, 8, 9). A 72 kDa band corresponding to Phospho-PAK4 + PAK6 + PAK7 (pSer474) was observed across the cell lines tested, which specifically gets competed with phosphopeptide and not with the nonphosphorylated peptide. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:5000 dilution). 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, MCF-7 cells were fixed and permeabilized for detection of endogenous Phospho-PAK4 + PAK6 + PAK7 pSer474 using Anti- Phospho-PAK4 + PAK6 + PAK7 pSer474 Recombinant Rabbit Polyclonal Antibody (Product # 711694, 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 Phospho-PAK4 + PAK6 + PAK7 pSer474 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 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of panels a, b and c clearly demonstrating nuclear localization of Phospho-PAK4 + PAK6 + PAK7 pSer474. Panel e) shows loss of signal by competition with the Phospho-PAK4 + PAK6 + PAK7 pSer474 phospho 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.