720070

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)

Flow cytometry (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

720070

Provider

Invitrogen Antibodies

Product name

H2AK5ac Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

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 nuclear enriched cell extracts (30 µg lysate) of HeLa (Lane1), HeLa treated with SAHA (0.5 uM/24 hrs) (Lane 2), HeLa treated with Sodium butyrate (5mM/24 hours) (Lane 3), HCT 116 (Lane 4), HCT 116 treated with SAHA (0.5 uM/24 hours) (Lane 5), HCT 116 treated with Sodium butyrate (5mM/24 hours) (Lane 6), Jurkat (Lane 7), Jurkat treated with SAHA (0.5 uM/24 hours) (Lane 8), Jurkat treated with Sodium butyrate (5mM/24 hours) (Lane 9) and K562 (Lane 10) and K562 treated with SAHA (0.5 uM/24 hrs) (Lane 11). The blot was probed with Anti-Histone H2AK5ac Rabbit Polyclonal Antibody (Product # 720070, 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 diln). A clear 17kDa 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 Novex® ECL Chemilumines

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence was performed on fixed and permeabilized HeLa cells for detection of Histone H2AK5ac using Anti-Histone H2AK5ac Rabbit Polyclonal Antibody (Product # 720070, 0.5 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:400). Panel a) shows representative cells that were stained for detection and localization of Histone H2AK5ac 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 Histone H2AK5ac. 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 Histone H2AK5ac was performed on Jurkat cells labeled with Anti-Histone H2AK5ac Rabbit Polyclonal Antibody (Product# 720070, 2-4 ug/ 1M cells) or with Rabbit isotype control and detected with Goat anti-Rabbit IgG (H+L) Superclonal™ 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 of 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer (4468770).

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Chromatin Immunoprecipitation (ChIP) was performed using Anti-Histone H2AK5ac Rabbit Polyclonal Antibody (Product # 720070, 5 µg) on sheared chromatin from 2 million HeLa cells treated with 5mM sodium butyrate for 24 hours using the "MAGnify ChIP system" kit (Product # 49-2024). Normal Rabbit IgG 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 cFOS region 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.