Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

HeLa cells were fixed in 100% methanol and permeabilized with 0.1% Triton X-100. Primary antibody, ab52484 at 1 : 2000 was incubated overnight at 4° C, followed by AlexaFluor® 488-conjugated Goat anti-mouse secondary antibody (ab150117) at 1/1000 dilution at RT for 45 min. ab179513 Anti-beta Tubulin, used as a counterstain at 1/200 dilution, was co-incubated with ab52484 overnight at 4° C, followed by Alexa Fluor® 594 Goat Anti-Rabbit secondary (ab150080) at 1/1000 dilution at RT for 45 min. Nucleus were visualized using DAPI.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

Flow cytometric analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling Histone H2B with ab52484 at 1/1000 dilution (0.1µg) (Red) compared with a Mouse monoclonal IgG (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti mouse IgG (Alexa Fluor® 488, ab150113) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

IHC image of Histone H2B staining in human breast carcinoma FFPE section, performed on a Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab52484, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

ICC/IF image of ab52484 stained human HeLa cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab52484, 1μg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

Nuclear staining in human breast cancer. The section was heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins. The section was incubated with ab52484 at 1 : 20000 (0.053 μg/ml) for 10 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. The immunostaining was performed on a Leica Biosystems BOND® RX instrument

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

Overlay histogram showing HeLa cells stained with ab52484 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab52484, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• ChIP

Lab

ChIP - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol*. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25 µg of chromatin, 2 µg of ab52484 (red), or 2 µg of mouse IgG1 ab18443 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

• IP

Lab

Immunoprecipitation - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

Histone H2B was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab52484 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab52484 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.  Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate  10 ug Lane 2 : ab52484 IP in HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate Lane 3 : Mouse monoclonal IgG (ab18443) instead of ab52484 in HeLa whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 32 seconds.

All lanes:

Immunoprecipitation - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484) at 1/30 dilution

All lanes:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 14 kDa

Observed band size: 15 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

Histone H2B was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Mouse monoclonal to Histone H2B and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab52484.
Secondary : Protein G-HRP at 1/500 dilution.
Band : 17kDa; Histone H2B; non specific bands - 26kDa : We are unsure as to the identity of this extra band.

All lanes:

Immunoprecipitation - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)

Predicted band size: 14 kDa

false

• ChIP

Supplier Data

ChIP - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25μg of chromatin, 5μg of ab52484 (blue), and 20μl 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

Nuclear staining in mouse lung. The section was heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins. The section was incubated with ab52484 (1 : 50000 (0.021 μg/ml)) for 10 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. The immunostaining was performed on a Leica Biosystems BOND® RX instrument

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

Flow cytometric analysis of NIH/3T3 (mouse embryonic fibroblast) cells labelling Histone H2B with ab52484 at 1/100 dilution (1µg) (Red) compared with a Mouse monoclonal IgG (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti mouse IgG (Alexa Fluor® 488, ab150113) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

NIH/3T3 cells were fixed in 100% methanol and permeabilized with 0.1% Triton X-100. Primary antibody, ab52484 at 1 : 2000 was incubated overnight at 4° C, followed by AlexaFluor® 488-conjugated Goat anti-mouse secondary antibody (ab150117) at 1/1000 dilution at RT for 45 min. ab179513 Anti-beta Tubulin, used as a counterstain at 1/200 dilution, was co-incubated with ab52484 overnight at 4° C, followed by Alexa Fluor® 594 Goat Anti-Rabbit secondary (ab150080) at 1/1000 dilution at RT for 45 min. Nucleus were visualized using DAPI.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

Nuclear staining in rat lung. The section was heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins. The section was incubated with ab52484 (1 : 50000 (0.021 μg/ml)) for 10 mins at room temperature, followed by anti-mouse IgG1 antibody (ab125913) for 8 mins during the LeicaDS9800 kit staining procedure. The immunostaining was performed on a Leica Biosystems BOND® RX instrument

• IP

Lab

Immunoprecipitation - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

Histone H2B was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast), whole cell lysate with ab52484 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab52484 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP) (ab131366) was used at 1/5000 dilution.  Lane 1 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10 μg Lane 2 : ab52484 IP in NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10 μg Lane 3 : Mouse monoclonal IgG (ab18443) instead of ab52484 in NIH/3T3 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 32 seconds.

All lanes:

Immunoprecipitation - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484) at 1/30 dilution

All lanes:

NIH/3T3 (mouse embryonic fibroblast), whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

• WB

Lab

Western blot - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

All lanes:

Western blot - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg

Lane 3:

MEF (mouse embryonic fibroblast (immortalized)), whole cell lysate at 20 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 20 µg

Lane 5:

Human heart tissue lysate at 20 µg

Lane 6:

Mouse heart tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/5000 dilution

Predicted band size: 14 kDa

Observed band size: 15 kDa

false

Exposure time: 3.25s

• WB

Supplier Data

Western blot - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

This image was generated using a
previous batch manufactured using hybridoma production method

All lanes:

Western blot - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484) at 5 µg/mL

Lane 1:

Marker

Lane 2:

Zebrafish brain homogenate at 20 µg

Lane 3:

Zebrafish heart homogenate at 10 µg

Lane 4:

Zebrafish liver homogenate at 10 µg

Lane 5:

Zebrafish skeletal muscle homogenate at 10 µg

Lane 6:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Mouse IgG – H&L – Pre-Adsorbed (HRP) at 1/6000 dilution

Predicted band size: 14 kDa

Observed band size: 17 kDa

true

Exposure time: 1min

• WB

Supplier Data

Western blot - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

All lanes:

Western blot - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484) at 1 µg/mL

Lane 1:

NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

Lane 2:

PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/5000 dilution

Predicted band size: 14 kDa

Observed band size: 17 kDa

true

Exposure time: 1min

• ICC/IF

AbReview37080****

Immunocytochemistry/ Immunofluorescence - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

This image was generated using a previous batch manufactured using hybridoma production method.

ab52484 staining Histone H2B in Fruit fly (Drosophila melanogaster) embryo cells by ICC/IF (Immunocytochemistry/immunofluorescence). Embryos were washed in 1x PBS + 0.1% Trition, cells were fixed with heat, and blocked with 10% BSA for 12 hours at 4°C. Samples were incubated with primary antibody (1/1000) for 24 hours. An Alexa Fluor®594-conjugated Rabbit anti-mouse IgG polyclonal (1/5000) was used as the secondary antibody.

This image is courtesy of an anonymous Abreview

• WB

Supplier Data

Western blot - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (AB52484)

All lanes:

Western blot - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484) at 5 µg/mL

Lane 1:

Histone H1 recombinant protein at 0.1 µg

Lane 2:

Histone H2A recombinant protein at 0.1 µg

Lane 3:

Histone H2B recombinant protein at 0.1 µg

Lane 4:

Histone H3 recombinant protein at 0.1 µg

Lane 5:

Histone H4 recombinant protein at 0.1 µg

Secondary

All lanes:

Rabbit polyclonal to Mouse IgG - H&L (HRP) at 1/3000 dilution

Predicted band size: 14 kDa

Observed band size: 17 kDa

false