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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [1]
- Immunocytochemistry [1]
- Flow cytometry [1]
- Other assay [1]
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- Product number
- 701799 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- H2BK15ac Recombinant Rabbit Monoclonal Antibody (7H17L5)
- Antibody type
- Monoclonal
- Antigen
- Synthetic peptide
- Description
- Since it is highly conserved across species, the antibody may react with many other species.
- Antibody clone number
- 7H17L5
- Concentration
- 0.5 mg/mL
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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).
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.
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).
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.
