720219

antibody from Invitrogen Antibodies

Targeting: RBPJ CBF1, IGKJRB, IGKJRB1, KBF2, RBP-J, RBPJK, RBPSUH, SUH

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

720219

Provider

Invitrogen Antibodies

Product name

RBPJ Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Other

Description

These Polyclonal antibodies are of rabbit origin developed by immunizing animals with proteins or peptides. The polyclonal antibody is purified by affinity purification from the rabbit sera generated after immunizing the rabbits with a specific type of protein or peptide. The purified antibody is tested for its functionality in various relevant research applications. The antibody is developed for Research Use Only and is non-hazardous or non-infectious in nature.

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 NTERA-2 (Lane 1), U-87 MG (Lane 2), A-431 (Lane 3), COLO 205 (Lane 4), HEK-293 (Lane 5) and U-937 (Lane 6). The blots were probed with Anti-CSL/RBPJ Rabbit Polyclonal Antibody (Product # 720219, 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 56 kDa band corresponding to CSL/RBPJ was observed across the cell lines tested. 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, HeLa cells were fixed and permeabilized for detection of endogenous CSL/ RBP J using CSL/RBP J Rabbit Polyclonal Antibody (Product # 720219, 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 CSL/ RBP J 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 demonstrating predominant nuclear localization of CSL/ RBP J. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Enrichment of endogenous CSL/RBP J protein at specific gene loci using Anti-CSL/RBP J Rabbit Polyclonal Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-CSL/RBP J Rabbit Polyclonal Antibody (Product # 720219, 3 µg) on sheared chromatin from 2 million HeLa cells using the MAGnify ChIP system kit (Product # 49-2024). Normal Rabbit IgG (1 µg) was used as a negative IP control. The purified DNA was analyzed by 7500 Fast qPCR system (Product # 4351106) with optimized PCR primer pairs for the promoters of the active HES1, HES5 promoter region used as positive control target genes, and the region of the inactive HES5 upstream, SAT2 satellite repeat, used as negative control target gene. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.