702266

antibody from Invitrogen Antibodies

Targeting: RPS6KA1 HU-1, MAPKAPK1, p90Rsk, RSK1

Western blot (Supportive data in Antibodypedia)

Antibody Data

Product number

702266

Provider

Invitrogen Antibodies

Product name

Phospho-RSK1 (Ser380) Recombinant Rabbit Monoclonal Antibody (13H23L1)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Monkey, Bovine, Mouse, Rat

Antibody clone number

13H23L1

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 HeLa, HeLa (Serum starved for overnight and treated with 25 ng/mL of TNFa for 20 min). The blots were probed with Anti-Phospho-RSK1 (Ser380) Recombinant Rabbit Monoclonal Antibody (Product # 702266, 1-2 µg/mL) (Lane 1, 2). To confirm the specificity of Phospho-RSK1 (Ser380), competition was performed with the phospopeptide (10 µg/mL) (Lane 3, 4) and non phospopeptide (10 µg/mL) (Lane 5, 6). A 90 kDa band corresponding to RSK1 (pS380) was observed in HeLa (with increase in expression upon treatment) when probed with the antibody alone or with non-phospho peptide competition. The blots were detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 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

Western Blot was performed using Anti-Phospho-RSK1 (Ser380) Recombinant Rabbit Monoclonal Antibody (13H23L1) (Product # 702266) and a 80 kDa band corresponding to Phosphorylated Ribosomal protein S6 kinase alpha-1 was observed. Nuclear enriched extracts (30 µg lysate) of HeLa (Lane 1), HeLa treated with PMA (Over-night serum starvation followed by treatment with 25 ng/mL PMA for 20 mins) (Lane 2) and HeLa treated with EGF (Over-night serum starvation followed by treatment with 100 nM EGF for 10 mins) (Lane 3) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The Blot was probed with the primary antibody (1 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Up-regulation in the expression of phosphorylated RSK1 was observed in the lanes treated with PMA and EGF as expected (https://doi.org/10.1038/onc.2012.472, doi: 10.1091/mbc.E11-07-0658).