701796-20UG

antibody from Invitrogen Antibodies

Targeting: H4C14 H4/n, H4F2, H4FN, HIST2H4, HIST2H4A

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

701796-20UG

Provider

Invitrogen Antibodies

Product name

H4K8ac Recombinant Rabbit Monoclonal Antibody (8H5L4), ChIP-Verified

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

8H5L4

Concentration

0.5 mg/mL

References

Submitted references

Interferon regulatory factor 1 and a variant of heterogeneous nuclear ribonucleoprotein L coordinately silence the gene for adhesion protein CEACAM1.

Dery KJ, Silver C, Yang L, Shively JE
The Journal of biological chemistry 2018 Jun 15;293(24):9277-9291

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (30 µg lysate) of HCT 116 (Lane1), HCT 116 treated with SAHA (0.5 uM/ 24 hours) (Lane 2), HCT 116 treated with sodium butyrate (5mM/ 24hours) (Lane 3), HeLa (Lane 4), HeLa treated with SAHA (0.5 uM/ 24 hours) (Lane 5), HeLa treated with sodium butyrate (5mM/ 24 hours) (Lane 6), K562 (Lane 7), K562 treated with SAHA (0.5 uM/ 24 hours) (Lane 8), Jurkat (Lane 9) and Jurkat treated with SAHA (0.5 uM/ 24 hours) (Lane 10). The blots were probed with Anti-Histone H4K8ac Recombinant Rabbit Monoclonal Antibody (Product # 701796 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 clear 17kDa band corresponding to Histone H4K8ac 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 Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (30 µg lysate) of HCT 116 (Lane1), HCT 116 treated with SAHA (0.5 uM/ 24 hours) (Lane 2), HCT 116 treated with sodium butyrate (5mM/ 24hours) (Lane 3), HeLa (Lane 4), HeLa treated with SAHA (0.5 uM/ 24 hours) (Lane 5), HeLa treated with sodium butyrate (5mM/ 24 hours) (Lane 6), K562 (Lane 7), K562 treated with SAHA (0.5 uM/ 24 hours) (Lane 8), Jurkat (Lane 9) and Jurkat treated with SAHA (0.5 uM/ 24 hours) (Lane 10). The blots were probed with Anti-Histone H4K8ac Recombinant Rabbit Monoclonal Antibody (Product # 701796 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 clear 17kDa band corresponding to Histone H4K8ac 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 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 H4K8ac using Anti-Histone H4K8ac Recombinant Rabbit Monoclonal Antibody (Product # 701796, 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 H4K8ac 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 H4K8ac. Panel e) represents control cells with no primary antibody to assess background.

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 H4K8Ac using Anti-Acetyl-Histone H4 (Lys8) Recombinant Rabbit Monoclonal Antibody: An array of the specific peptide and other relevant peptides when tested using Anti-Acetyl-Histone H4 (Lys8) Recombinant Rabbit Monoclonal Antibody (Product # 701796), showed that the Histone H4K8Ac modification was specifically recognized by the antibody.

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 H4K8Ac using Anti-Acetyl-Histone H4 (Lys8) Recombinant Rabbit Monoclonal Antibody: An array of the specific peptide and other relevant peptides when tested using Anti-Acetyl-Histone H4 (Lys8) Recombinant Rabbit Monoclonal Antibody (Product # 701796), showed that the Histone H4K8Ac modification was specifically recognized by the antibody.

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 H4K8ac Recombinant Rabbit Monoclonal Antibody (Product # 701796) and H4K8ac was enriched compared to the other histone modifications in the SNAP-ChIP™ K-AcylStat Panel (Product # A47358).