711129

antibody from Invitrogen Antibodies

Targeting: RB1 OSRC, PPP1R130, RB

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

711129

Provider

Invitrogen Antibodies

Product name

Phospho-Rb (Ser807, Ser811) Recombinant Polyclonal Antibody (13HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Monkey, Bovine and Pig.

Antibody clone number

13HCLC

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on Nuclear enriched extracts (30 µg lysate) of LNCaP (Lane 1), LNCaP treated with Nocodazole (83 nM for 24hrs) (Lane 2), K-562 (Lane 3), K-562 treated with Nocodazole (83 nM for 24hrs) (Lane 4), MCF-7 (Lane 5) and MCF-7 treated with Nocodazole (83 nM for 24hrs) (Lane 6). The blots were probed with Anti-Rb (pS 807/811) Recombinant Rabbit Polyclonal Antibody (Product # 711129, 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). A 110 kDa band corresponding to Rb (pS 807/811) was observed across the cell lines tested and * non specific bands also observed. 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).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, LNCaP cells were fixed and permeabilized for detection of endogenous Rb (pS 807/811) using Rb (pS 807/811) Recombinant Rabbit Polyclonal Antibody (Product # 711129, 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 Rb (pS 807/811) 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 localization of Rb (pS 807/811). Panel e) shows loss of signal by competition with the Rb (pS 807/811) phosphopeptide, demonstrating antibody specificity, and panel f) demonstrates no competition with the non-phospho peptide. Panel g) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.