720006

antibody from Invitrogen Antibodies

Targeting: RELA NFKB3, p65

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Antibody Data

Product number

720006

Provider

Invitrogen Antibodies

Product name

Phospho-NFkB p65 (Ser536) Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Monkey, Rat, Mouse, Pig, Bovine, Rabbit and Dog.

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (50 µg lysate of Jurkat (lane 1), Jurkat treated with TNF-alpha (50 ng/mL/20 min) (lane 2) and Jurkat treated with Calyculin A (50 ng/mL/20 min) (lane3). The blots were probed with Anti-NF-kappa-B p65 (pS536)Rabbit Polyclonal Antibody (Product # 720006, 2-3 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 clear 65 kDa band corresponding to NF-kappa-B p65 (pS536)was observed across cell lines tested according to the treatment. 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

Immunofluorescence was performed on fixed and permeabilized Jurkat cells treated with Calyculin A (50 ng/mL/20 min) for detection of NF-kappa-B p65 (pS 536) using Anti-NF-kappa-B p65 (pS536)Rabbit Polyclonal Antibody (Product # 720006, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 0.4 µg/mL, 1:2500). Panel a) shows representative cells that were stained for detection and localization of NF-kappa-B p65 (pS536) protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938, 1:50). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 594 Phalloidin (Product # A12381, 1:200). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of NF-kappa-B p65 (pS 536). Panel e) represents control cells with no primary antibody to assess background.

Flow cytometry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Flow Cytometry analysis of NF-kappa-B p65 [pS536] was performed on Jurkat cells treated with Calyculin A (50ng/ml/ 20 min) cells labeled with Anti-NF-kappa-B p65 [pS536] Rabbit Polyclonal Antibody (Product# 720006, 2-4 ug/ 1M cells) or with rabbit isotype control and detected with Goat anti-Rabbit IgG (H+L) Superclonalª Secondary Antibody, Alexa Fluor¨ 488 conjugate (Product # A27034, 0.4 ug/ml, 1:2500) as represented by the red and pink histograms respectively. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control. A representative 10,000 cells were acquired and analyzed for each sample using an Attune¨ Acoustic Focusing Cytometer (4468770).