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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [1]
- Immunocytochemistry [1]
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- Product number
- 711694 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-PAK4/6/7 (Ser474) Recombinant Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- This antibody is predicted to react with Monkey, Horse, Rat
- Concentration
- 0.5 mg/mL
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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)
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.
