Anti-Histone H4 antibody [EPR16599] - ChIP Grade

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 antibody [EPR16599] - ChIP Grade (AB177840)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling Histone H4 with ab177840 at 1/2000 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nucleus staining on glandular epithelium of Human colon tissue is observed. Counter stained with Hematoxylin.

Negative control : PBS instead of primary antibody; secondary antibody is prediluted Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Histone H4 antibody [EPR16599] - ChIP Grade (AB177840)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa cells labeling Histone H4 with ab177840 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/400 dilution (green). Nuclear staining on HeLa cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows :
1. ab177840 at 1/100 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

• ChIP

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ChIP - Anti-Histone H4 antibody [EPR16599] - ChIP Grade (AB177840)

Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with 0.75% formaldehyde for 10min. The ChIP was performed with 25μg of chromatin, 2μg of ab177840 (blue), and 20μl of Anti rabbit IgG sepharose beads. 2μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (SYBR approach). Primers are located in the first kb of the transcribed region.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 antibody [EPR16599] - ChIP Grade (AB177840)

Immunohistochemical analysis of paraffin-embedded Mouse pancreas tissue labeling Histone H4 with ab177840 at 1/2000 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nucleus staining on glandular epithelium of mouse pancreas tissue is observed. Counter stained with Hematoxylin.

Negative control : PBS instead of primary antibody; secondary antibody is prediluted Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 antibody [EPR16599] - ChIP Grade (AB177840)

Immunohistochemical analysis of paraffin-embedded Rat cerebral cortex tissue labeling Histone H4 with ab177840 at 1/2000 dilution, followed by prediluted Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining on neuron cells of cerebral cortex tissue is observed. Counter stained with Hematoxylin.

Negative control : PBS instead of primary antibody; secondary antibody is prediluted Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

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Western blot - Anti-Histone H4 antibody [EPR16599] - ChIP Grade (AB177840)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Histone H4 antibody [EPR16599] - ChIP Grade (ab177840) at 1/5000 dilution

Lane 1:

HeLa whole cell lysates at 10 µg

Lane 2:

NIH/3T3 whole cell lysates at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

true

Exposure time: 3min

• WB

Lab

Western blot - Anti-Histone H4 antibody [EPR16599] - ChIP Grade (AB177840)

Lanes 1-5 : 1% BSA blocking buffer

Lanes 6-10 : 3% Milk blocking buffer

We recommend using 3% milk as the blocking agent for Western blot.

All lanes:

Western blot - Anti-Histone H4 antibody [EPR16599] - ChIP Grade (ab177840) at 1/1000 dilution

Lanes 1 and 6:

Histone H1 Recombinant Protein at 0.1 µg

Lanes 2 and 7:

Histone H2A Recombinant Protein at 0.1 µg

Lanes 3 and 8:

Histone H2B Recombinant Protein at 0.1 µg

Lanes 4 and 9:

Histone H3.1 Recombinant Protein at 0.1 µg

Lanes 5 and 10:

Histone H4 Recombinant Protein at 0.1 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (H+L), Peroxidase Conjugated at 1/50000 dilution

true

Exposure time: 4min

• WB

Supplier Data

Western blot - Anti-Histone H4 antibody [EPR16599] - ChIP Grade (AB177840)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Histone H4 antibody [EPR16599] - ChIP Grade (ab177840) at 1/1000 dilution

All lanes:

Drosophila whole lysates at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

true

Exposure time: 3min

• PepArr

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Peptide Array - Anti-Histone H4 antibody [EPR16599] - ChIP Grade (AB177840)

ab177840 was tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).
Circle area represents affinity between the antibody and a peptide : all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.
The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, is available under the Product Protocol section.