b'AntibodiesH3.3 G34W oncohistone mutant monoclonal antibody for cancer researchOncohistones are mutant histone proteins with oncogenic features.G34W mutations in histone H3.3 influence pre-mRNA processing and Single amino acid substitutions in the tails of core histones often leadalternative splicing and are proliferative and infiltrative in nature. They to the formation of brain, cartilage, or bone tumors. Mutations due tocontribute to the tumorigenic process that leads to overgrowth and substitution of glycine (G) 34 by tryptophan (W), valine (V), argininebone destruction in GCTB.(R), or leucine (L) in histone H3.3 are responsible for the majority of giant cell bone tumors. Giant cellrich primary bone tumors areThe greatest challenge in identifying giant cellrich bone tumors locally aggressive subarticular tumors characterized by the presenceis working with limited biopsy samples, so it is crucial to have a of prominent osteoclasts or osteoclast-like giant cells. The G34Wspecific antibody. Highly specific and sensitive Invitrogen H3.3 G34W mutation is the most common variant and is found in 90% of patientsoncohistone mutant recombinant rabbit monoclonal antibody (Cat. No. diagnosed with giant cell tumors in bone (GCTB). 703836) has been verified for western blotting and immunofluorescence applications with Invitrogen SNAP-ChIP technology. It can be a powerful tool for detecting the H3.3 G34W mutation in GCTB (Figure 5).A B H3.3 G34W DAPI F-Actin Figure 5. Chromatin immunoprecipitation (ChIP) demonstrating the specificity Transfected of H3.3 G34W oncohistone antibody. (A) Western blot obtained with H3.3 G34W oncohistone mutant recombinant rabbit monoclonal antibody. A 17 kDa band corresponding to histone H3.3 G34W was observed with HEK293T cells that overexpressed the mutant protein, confirming the specificity of the H3.3 G34W antibody. 1 2 A B C Lane 1: whole-cell extract (1% SDS) (30 g lysate) from untransfected control cells. Lane Composite H3.3 G34W No primary antibody 2: whole-cell extract of HEK293T cells transfected with a histone H3.3 G34W construct. 260 Untransfected The membrane was probed with the primary antibody (1:5,000 dilution) and detected by 160110 chemiluminescence with Invitrogen goat anti-rabbit IgG (H+L) Superclonal Recombinant 80 Secondary Antibody, HRP conjugate (Cat. No. A27036; 1:4,000 dilution) using the 60 Invitrogen iBright FL1500 Imaging System (Cat. No. A44115). Chemiluminescence 50 detection was performed using Thermo Scientific Pierce ECL Western Blotting Substrate 40 D E F (Cat. No. 32106). (B) For immunofluorescence analysis, HEK293 cells transfected with 30H3.3 G34W a histone H3.3 G34W construct were fixed and permeabilized with 0.5% Triton X-100 20 ~17 kDa C 120 100 surfactant for detection of histone H3.3 G34W using (panel A, green) H3.3 G34W 15 oncohistone mutant recombinant rabbit monoclonal antibody (Cat. No. 703836; 1:100 100 10 Enrichment (% input) dilution) and Invitrogen donkey anti-rabbit IgG (H+L) highly cross-adsorbed secondary Specicity (% target)10 80 antibody, Alexa Fluor Plus 488 conjugate (Cat. No. A32790; 1:2,000). Nuclei (panel B, 60 1 blue) were stained using Invitrogen ProLong Diamond Antifade Mountant with DAPI 3.5 (Cat. No. P36962). F-actin (panel C, red) was stained using Invitrogen Rhodamine 40 Phalloidin (Cat. No. R415, 1:300). Panel D is a merged image showing nuclear localization. 0.120 Panel E shows untransfected control cells, and panel F shows cells without primary GAPDH 0.01 antibody to assess background. The images were captured at 60x magnification. (C) 0HEK293T cells Antibody specificity was demonstrated using SNAP-ChIP spike-in technology, which+ overexpressing H3.3 G34W H3.3 K4M K9M K27M K36M G34R G34V G34WAb eciencyutilizes DNA-barcoded nucleosomes that are spiked into a sample of isolated nuclei or enriched chromatin. By following ChIP with an antibody that targets a specific histone modification or mutation, the efficiency of antibody enrichment can be determined via qPCR. SNAP-ChIP spike-in was performed using Invitrogen OncoStat Panel SNAP-ChIP Spike-in (Cat. No. A47343) and H3.3 G34W oncohistone mutant recombinant rabbit monoclonal antibody. Histone H3.3 G34W was enriched relative to the other histone mutations in the panel.Learn more about Invitrogen antibody validation at thermofisher.com/antibodyvalidation18 Inside the Cell//Issue No. 11Contents '