701799

antibody from Invitrogen Antibodies

Targeting: H2BC11 H2B/r, H2BFR, HIST1H2BJ

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Flow cytometry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

701799

Provider

Invitrogen Antibodies

Product name

H2BK15ac Recombinant Rabbit Monoclonal Antibody (7H17L5)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

7H17L5

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 analysis was performed on whole cell extracts (30 µg of nuclear enriched lysate) of HCT 116 (Lane1), HCT 116 treated with SAHA (0.5 uM/24 hrs) (Lane 2), HCT 116 treated with sodium butyrate (5mM/24 hrs) (Lane 3), HeLa (Lane 4), HeLa treated with SAHA for (0.5 uM/24 hrs) (Lane 5), HeLa treated with sodium butyrate (5mM/ 24hrs) (Lane 6), Jurkat (Lane 7), Jurkat treated with SAHA (0.5 uM/24 hrs) (Lane 8) and Jurkat treated with sodium butyrate (5mM/24 hrs) (Lane 9). The blots were probed with Anti-Histone H2BK15 Recombinant Rabbit Monoclonal Antibody (Product # 701799 0.5-1 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 and 14 kDa band corresponding to the 2 abundant isoforms of Histone H2BK15 was observed across cell lines according to the treatment. 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 primary and secondary antibody following blocking with 5% nonfat 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

Immunofluorescence was performed on MCF7 cells treated with SAHA (0.5 uM/18 hours). These cells were permeabilized and fixed for detection of Histone H2BK15ac using Anti-Histone H2BK15ac Recombinant Rabbit Monoclonal Antibody (Product # 701799, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 0.4 µg/mL, 1:2500). Panel a) shows representative cells that were stained for detection and localization of Histone H2BK15ac protein (green), Panel b) is stained for Nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938, 1:50). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 594 Phalloidin (Product # A12381, 1:200). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of Histone H2BK15ac. 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 H2BK15ac was performed on Jurkat cells labeled with ABfinityª Anti-Histone H2BK15ac Recombinant Rabbit Monoclonal Antibody (Product# 701799, 2-4 ug/ 1M cells) or with rabbit isotype control and detected with Goat anti-Rabbit IgG (H+L) Superclonalª 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).

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Antibody specificity for modified targets can be established using peptide arrays by quantifying detection of the target protein along with closely related proteins. Peptide array of Histone H2BK15Ac using Anti-Acetyl-Histone H2B (Lys15) Recombinant Rabbit Monoclonal Antibody: An array of the specific peptide and other relevant peptides when tested using Anti-Acetyl-Histone H2B (Lys15) Recombinant Rabbit Monoclonal Antibody (Product # 701799), showed that the Histone H2BK15Ac modification was specifically recognized by the antibody.