702933

antibody from Invitrogen Antibodies

Targeting: H3-3A H3.3A, H3F3, H3F3A

Western blot (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

702933

Provider

Invitrogen Antibodies

Product name

H3.3 K9M oncohistone mutant Recombinant Rabbit Monoclonal Antibody (22H22L9), ChIP-Verified

Antibody type

Monoclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Mouse, Rat. Recombinant human histone H3.3 (H3F3A, H3.3A) containing a methionine at position 9.

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

22H22L9

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 was performed using Anti-H3.3 K9M Rabbit Recombinant Monoclonal Antibody (Product # 702933) and a 17 kDa band corresponding to H3.3 K9M was observed in histone H3.3 K9M overexpressing HEK-293T cells confirming the specificity of H3.3 K9M antibody. Modified whole cell extracts (1% SDS) (30 µg lysate) from control (untransfected) (Lane 1), FLAG-tagged histone H3.3 K9M overexpressing HEK-293T cells (Lane 2) were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0341BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:500 dilution) and detected by chemiluminescence Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (Product # A27036, 1:4000 dilution). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

OncoStat Panel, SNAP-ChIP™ Spike-in (Product # A47343), a proprietary technology developed by EpiCypher™ was used to analyze the performance of H3.3 K9M antibody (Product # 702933) in ChIP. SNAP-ChIP panels consist of a pool of DNA-barcoded recombinant nucleosomes harboring histone mutations that are spiked-in to a ChIP reaction to assess efficiency and specificity of the antibody. The OncoStat panel includes an unmodified H3.3 control plus nucleosomes containing H3.3 mutations (K4M, K9M, K27M, G34R, G34V, G34W and K36M) as shown on the x-axis. Recovery of each unique DNA-barcoded nucleosome is quantified to determine how much of each PTM is immunoprecipitated in the ChIP reaction (for more information see reference ). H3.3 K9M antibody was tested in native ChIP with 3 µg K-562 cell chromatin and 3 µg antibody. Specificity (left Y-axis) was determined by qPCR to each modified nucleosome in the panel (X-axis). Black bar represents antibody efficiency (right Y-axis; log scale) and indicates percentage of the barcoded nucleosome target immunoprecipitated relative to Input. All bars represent mean ± SEM.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Antibody specificity was demonstrated by detection of enrichment of the targeted histone modification using SNAP-ChIP™ Spike-in, a proprietary technology developed by EpiCypher™. SNAP-ChIP™ spike-in was performed using H3.3 K9M Recombinant Rabbit Monoclonal Antibody (Product # 702933) and H3.3 K9M was enriched compared to the other histone modifications in the SNAP-ChIP™ OncoStat Panel (Product # A47343).