Anti-Histone H3 antibody - ChIP Grade (ab1791)

Western blot - Histone H3 antibody - ChIP Grade (ab1791)

All lanes : Anti-Histone H3 antibody - ChIP Grade (ab1791) at 1/1000 dilution

Lane 1 : A431 Whole Cell Lysate
Lane 2 : Jurkat Whole Cell Lysate
Lane 3 : 293 Whole Cell Lysate
Lane 4 : A431 Whole Cell Lysate with Histone H3 peptide (ab12149) at 1 µg/ml
Lane 5 : Jurkat Whole Cell Lysate with Histone H3 peptide (ab12149) at 1 µg/ml
Lane 6 : 293 Whole Cell Lysate with Histone H3 peptide (ab12149) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG - H&L (HRP) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 15 kDa
Observed band size : 17 kDa (why is the actual band size different from the predicted?)


Exposure time : 10 seconds

ChIP - Histone H3 antibody - ChIP Grade (ab1791)

Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 µg of chromatin, 2 µg of ab1791 (blue), and 20 µl of protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 antibody - ChIP Grade (ab1791)

Hela cells cultured on coverslips were fixed with 4% paraformaldehyde and then stained with ab1791 (green) at a working dilution of 1/500. The DNA stained with DAPI is shown in red. (100x magnification).

Kirk McManus, University of British Columbia

ChIP - Histone H3 antibody - ChIP Grade (ab1791)

Chromatin from Xenopus laevis oocytes was prepared according to the Abcam X-ChIP protocol. Oocytes were fixed with formaldehyde for 10 min. The ChIP was performed with 25 mg of chromatin, 3 mg of ab7834 (anti-H3, light blue) and 3 µg of ab1791 (anti-H3, dark blue), and 20 ml of Protein A/G sepharose beads. A non-specific antibody was used as a control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach).

Flow Cytometry - Histone H3 antibody - ChIP Grade (ab1791)

 ab1791 staining mouse embryonic stem cells by flow cytometry (gated on all living cells). The cell colonies were trypsinized and incubated with the antibody 1ug/1.5 x 10⁵cells in a permeabilization buffer. A PE conjugated goat anti-rabbit antibody was used as the secondary.

This image is courtesy of an Abreview submitted by Prof Albrecht Müller

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H3 antibody - ChIP Grade (ab1791)

ab1791 staining rat testes tissue sections by IHC-P.  Sections were formaldehyde fixed and subjected to heat mediated antigen retrieval prior to blocking with 5% serum for 30 minutes at 22°C.  The primary antibody was diluted 1/400 and incubated with the sample for 30 minutes at 22°C.  A HRP-conjugated goat anti-rabbit antibody diluted 1/400 was used as the secondary.

This image is courtesy of an anonymous Abreview

Western blot - Histone H3 antibody - ChIP Grade (ab1791)

All lanes : Anti-Histone H3 antibody - ChIP Grade (ab1791) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab7179)
Lane 3 : Drosophila embryo nuclear extract (from melanogaster embryos 0-12Hr)
Lane 4 : S.cerevisiae (Y190) Whole Cell Lysate
Lane 5 : S.pombe Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Performed under reducing conditions.

Predicted band size : 15 kDa
Observed band size : 17 kDa (why is the actual band size different from the predicted?)


ab1791 is tested in western blot on a range of species.  We recommend loading higher amounts of protein (20-30ug) to increase the signal in yeast lysates

Immunocytochemistry/ Immunofluorescence - Histone H3 antibody - ChIP Grade (ab1791)

ICC/IF image of ab1791 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab1791, 1µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 5% PFA fixed (5 min) Hek293, HepG2 and MCF7 cells at 1µg/ml, and in 4% PFA fixed (10 min) HeLa and Hek293 cells at 1µg/ml.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H3 antibody - ChIP Grade (ab1791)

ab1791 staining Histone H3 in human benign nevi tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed. Samples were then blocked with 2.5% normal horse serum for 20 minutes followed by incubation with the primary antibody at a 1/200 dilution for 16 hours at 4ºC. An undiluted HRP-conjugated rabbit polyclonal was used as the secondary antibody.

Image courtesy of an anonymous Abreview.

Western blot - Histone H3 antibody - ChIP Grade (ab1791)

All lanes : Anti-Histone H3 antibody - ChIP Grade (ab1791) at 1/10000 dilution

Lane 1 : HeLa nuclear lysate
Lane 2 : HeLa nuclear lysate
Lane 3 : HeLa nuclear lysate

Lysates/proteins at 2 µg per lane.

Secondary
HRP conjugated donkey polyclonal at 1/2000 dilution
developed using the ECL technique

Predicted band size : 15 kDa


Exposure time : 5 seconds

Image courtesy of Dr Roman Hudec by Abreview.

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 antibody - ChIP Grade (ab1791)

ICC/IF image of ab1791 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab1791, 1µg/ml) overnight at +4ºC. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Electron Microscopy - Anti-Histone H3 antibody - ChIP Grade (ab1791)

Histone H3 immunogold detection in HeLa cells.
Ulltra-thin sections of paraformaldehyde-fixed, Lowicryl K4M embedded cells were incubated sequentially with ab1791 antibody and an anti-rabbit antibody coupled to 10 nm gold particles. Notice absence of chromatin within Interchromatin Granules (IG), a reservoir of spliceosomal snRNPs, and high labeling of patches of condensed chromatin close to the nuclear envelope (arrows). Nu: nucleus, Cyt: cytoplasm.

Sample : human
Type : HeLa cells
Fixative : either paraformaldehyde 4% or 1.6% glutaraldehyde in 0.1M Millonig’s buffer.
Embedding : in Lowicryl 4KM at -20°C under UV.
Ultrathin-sections deposited on formvar-coated carbonated EM-grids.
Blocking step: 5% BSA for 30 seconds at RT.
Primary antibody: Ab1791 diluted 1/50 in PBS, for 1h at RT.
Secondary antibody: BBI International anti-rabbit antibody coupled to 10 nm gold particles

Rating: excellent

Additional comments: A highly specific antibody working similarly on thin-sections of glutaraldehyde or paraformaldehyde-fixed cells. Ideal for testing antigeneicity, for detecting micronuclei, chromatin content of nuclear organelles or bodies…

Western blot - Histone H3 antibody - ChIP Grade (ab1791)

Predicted band size : 15 kDa

John E. Mueller and J. Ruth German (Mary Bryk lab)