711262

antibody from Invitrogen Antibodies

Targeting: GSK3B

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

711262

Provider

Invitrogen Antibodies

Product name

Phospho-GSK3 alpha/beta (Tyr279, Tyr216) Recombinant Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Monkey, rabbit and horse.

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 3T3-L1 (Lane 1), 3T3-L1 treated with Insulin (100nM-10min) (Lane 2), A549 (Lane 3) and A549 treated with Insulin (100nM-10min) (Lane 4). The blots were probed with Anti-GSK3a (pY279) + GSK3b (pY216) Recombinant Rabbit Polyclonal Antibody (Product # 711262, 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 46 kDa and 51 kDa band corresponding to GSK3a (pY279)+GSK3b (pY216) was observed. To confirm the specificity of Antibody, competition was performed with the phosphopeptide (10 µg/mL) as shown in the corresponding blot on the right. The phosphopeptide competes with the antibody and prevents it from binding to the target protein. 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 GSK-3 alpha (pY279) + GSK3 beta (pY216) using Anti-GSK-3 alpha (pY279) + GSK3 beta (pY216) Recombinant Rabbit Polyclonal Antibody (Product # 711262, 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 GSK-3 alpha (pY279) + GSK3 beta (pY216) 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 GSK-3 alpha (pY279) + GSK3 beta (pY216). Antibody specificity was demonstrated by competition with the GSK-3 alpha (pY279) + GSK3 beta (pY216) phospho peptide, which results in loss of signal in panel e). Panel f) shows no competition with the non-phospho peptide. The images were captured at 60X magnification