- 702305 - Invitrogen Antibodies TBC1D4 antibody

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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), HeLa treated with Insulin (100 nM, for 10 min) (Lane 2), HEK-293 (Lane 3) and HEK-293 treated with Insulin (100 nM, for 10 min) (Lane 4). The blots were probed with Anti-phospo-AS160 (pS558) Recombinant Rabbit Monoclonal Antibody (Product # 702305, 1 µg/mL). A 160 kDa band corresponding to AS160 (pS558) was observed across the cell line tested. 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® 10% Bis-Tris gel (Product # NP0301BOX), 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: Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HEK-293 (Lane 1) and HEK-293 treated with Insulin (100 nM, for 10 min) (Lane 2). The blots were probed either, with Anti-Phospho-Phospho-AS160 (Ser558) Recombinant Rabbit Monoclonal Antibody (Product # 702305, 1 µg/mL). To confirm the specificity competition was performed with the phospopeptide (10 µg/mL) (Lane 3 & 4) and non phospopeptide (10 µg/mL) (Lane 5 & 6). A 160 kDa band corresponding to Phospho-AS160 (Ser558) 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 wet transfer method. 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 HT-29 cells were fixed and permeabilized for detection of endogenous AS160 (Ps588) using Anti-Phospho-AS160 (Ser588) Recombinant Rabbit Monoclonal Antibody (Product # 702305, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Detection and localization of Phospho-AS160 (Ser588) (green) in the cytoplasm can be clearly observed in cells treated with IGF (100 ng/mL, 15 min) as compared to untreated cells. Antibody specificity was demonstrated by competition with the Phospho-AS160 (Ser588) phospho peptide, which results in loss of signal. No competition was observed with the non-phospho peptide. The images were captured at 60X magnification.

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