710830

antibody from Invitrogen Antibodies

Targeting: H2AC11 H2A.1b, H2A/p, H2AFP, HIST1H2AG, pH2A/f

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

710830

Provider

Invitrogen Antibodies

Product name

H2AK5ac Recombinant Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Rat, Pig, Dog and Cat.

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on acid extracts (30 µg lysate) of HEK-293 (Lane1), HEK-293 treated with Sodium butyrate (0.5 µM/18 hrs) (Lane 2), HeLa (Lane 3) and HeLa treated with Sodium butyrate (0.5 µM/18 hrs) (Lane 4). The blots were probed with Anti-Histone H2AK5ac Recombinant Rabbit Polyclonal Antibody (Product # 710830, 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 17 kDa band corresponding to Histone H2AK5ac was observed across 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 H2AK5ac using H2AK5ac Recombinant Rabbit Polyclonal Antibody (Product # 710830, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Detection and localization of H2AK5ac (green) in the nucleus can be clearly observed in cells treated with suberoylanilide hydroxamic acid (SAHA, 0.5 uM, 18 h) as compared to untreated cells. Antibody specificity was demonstrated by competition with the H2AK5ac peptide, which results in loss of signal. No competition was observed with the non-.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Enrichment of endogenous H2AK5ac protein at specific gene loci using Anti-Acetyl Histone H2A (Lys5) Recombinant Rabbit Polyclonal Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-Histone H2AK5ac Recombinant Rabbit Polyclonal Antibody (Product # 710830, 5 µg) on sheared chromatin from 2 million Jurkat cells treated with 5mM sodium butyrate for 24 hours 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 BActin, and cFOS genes, used as positive control target gene, and the region of the inactive 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.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Cross-reactivity ELISA for Anti-Histone H2AK5ac Recombinant Rabbit Polyclonal Antibody (Product # 710830) was performed against H2AK5ac and H2AK5-non ac by coating at 1 æg/mL. Antibodies at 1 æg/mL were added and detected using Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP conjugate (Product # G-21234, 1:5000 dilution). The plate was developed using Stabilized Chromogen, TMB (Product # SB02).