702448

antibody from Invitrogen Antibodies

Targeting: BRD4 CAP, HUNK1, HUNKI, MCAP

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

702448

Provider

Invitrogen Antibodies

Product name

BRD4 Recombinant Rabbit Monoclonal Antibody (14H4L4)

Antibody type

Monoclonal

Antigen

Other

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

14H4L4

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.

References

Submitted references

BRD4S interacts with viral E2 protein to limit human papillomavirus late transcription.

Yigitliler A, Renner J, Simon C, Schneider M, Stubenrauch F, Iftner T
Journal of virology 2021 May 10;95(11)

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 K-562 (Lane 1) and HEL 92.1.7 (Lane 2). The blots were probed with Anti-BRD4 Recombinant Rabbit Monoclonal Antibody (Product # 702448, 1 µg/mL). Isoforms of 200, 152 and 88 kDa bands corresponding to BRD4 was observed across the cell lines tested. 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® 10% Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane by 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, HeLa cells were fixed and permeabilized for detection of endogenous BRD4 using Anti- BRD4 Recombinant Rabbit Monoclonal Antibody (Product # 702448, 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 BRD4 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 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating Nuclear localization of BRD4. 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 BRD4 protein at specific gene loci using Anti-BRD4 Recombinant Rabbit Monoclonal Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-BRD4 Recombinant Rabbit Monoclonal Antibody (Product # 702448, 5 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 IL6, INOS regions used as positive control target gene, and the region of the 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.