- 701796 - Invitrogen Antibodies H4C14 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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.

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 (Heavy Chain) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow Cytometry analysis of Histone H4K8ac was performed on Jurkat cells labeled with ABfinityª Anti-Histone H4K8ac Recombinant Rabbit Monoclonal antibody (Product# 701796, 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)
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Experimental details: The specificity of the antibody, Anti-Acetyl-Histone H4 (Lys8) Recombinant Rabbit Monoclonal Antibody (Product # 701796, 0.25 µg/mL) to Histone H4K8Ac peptide was confirmed using MODified™ Histone Peptide Array (Active Motif, 13001). Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 1:4000 dilution) was used as the secondary antibody. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106). The dot blot data obtained (lower panel) was analyzed using Array Analyze software as per the manufacturer's instructions (top panel).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: K-AcylStat Panel, SNAP-ChIP™ Spike-in (Product # A47358), a proprietary technology developed by EpiCypher™ was used to analyze the performance of H4K8ac antibody (Product # 701796) in ChIP. SNAP-ChIP panels consist of a pool of DNA-barcoded recombinant nucleosomes harboring histone PTMs that are spiked-in to a ChIP reaction to assess efficiency and specificity of the antibody. The K-AcylStat panel includes an unmodified control plus nucleosomes containing histones with single acylations (acetylation, butrylation, or crotonylation) and combinatorial PTM configurations (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). H4K8ac 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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).

701796