Histone H3 Antibody (1B1-B2) - BSA Free

Novus Biologicals | Catalog # NBP2-36468

Novus Biologicals

Key Product Details

Validated by

Knockout/Knockdown

Species Reactivity

Validated:

Human, Mouse, Rat

Cited:

Human

Applications

Validated:

Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, Immunocytochemistry/ Immunofluorescence, Chromatin Immunoprecipitation (ChIP), Single Cell Western

Cited:

Western Blot

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG3 Kappa Clone # 1B1-B2

Format

BSA Free
View all Histone H3 Primary Antibodies »

Product Specifications

Immunogen

This Histone H3 antibody (1B1-B2) was raised against synthetic peptide corresponding to the C-terminus of human Histone H3.

Localization

Nucleus. Chromosome.

Clonality

Monoclonal

Host

Mouse

Isotype

IgG3 Kappa

Theoretical MW

15 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for Histone H3 Antibody (1B1-B2) - BSA Free

Chromatin Immunoprecipitation: Histone H3 Antibody (1B1-B2) [NBP2-36468] - Chromatin from one million formaldehyde cross-linked HeLa cells was precipitated using 2 ug of NBP2-36468 and 25 ul of magnetic IgG beads, using standard ChIP methods. A similar sample containing no antibody was included as a negative control. Immunoprecipitated DNA was quantified using quantitative real-time PCR and SYBR green dye, then normalized to the non-precipitated input chromatin. Representative target genes from active, inactive and heterochromatic regions of the genome show amplification, indicative of the presence of Histone H3.
Western Blot: Histone H3 Antibody (1B1-B2)BSA Free [NBP2-36468]

Western Blot: Histone H3 Antibody (1B1-B2)BSA Free [NBP2-36468]

Western Blot: Histone H3 Antibody (1B1-B2) [NBP2-36468] - Western blot image of Anti-Histone H3 antibody (clone 1B1-B2). Whole cell protein from HeLa (lane 1), NIH-3T3 (lane 2) and PC12 (lane 3) were separated by 4-15% SDS-PAGE and transfered to PVDF membrane. The membrane was probed with purified Anti-Histone H3 antibody at 2 ug/ml and detected with an HRP conjugated anti-mouse secondary using chemiluminescence. Observed molecular weight is ~15 kDa.

Immunohistochemistry-Paraffin: Histone H3 Antibody (1B1-B2) [NBP2-36468]

Immunohistochemistry-Paraffin: Histone H3 Antibody (1B1-B2) [NBP2-36468] - Analysis of Histone H3 in human breast cancer using DAB with hematoxylin counterstain.
Immunocytochemistry/ Immunofluorescence: Histone H3 Antibody (1B1-B2) - BSA Free [NBP2-36468]

Immunocytochemistry/ Immunofluorescence: Histone H3 Antibody (1B1-B2) - BSA Free [NBP2-36468]

Immunocytochemistry/Immunofluorescence: Histone H3 Antibody (1B1-B2) [NBP2-36468] - HeLa cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton-X100. The cells were incubated with anti-Histone H3 (1B1-B2) NBP2-36468 at a 1:200 dilution overnight at 4C and detected with an anti-mouse Dylight 488 (Green) at a 1:500 dilution. Actin was detected with Phalloidin 568 (Red) at a 1:200 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.
Western Blot: Histone H3 Antibody (1B1-B2)BSA Free [NBP2-36468]

Western Blot: Histone H3 Antibody (1B1-B2)BSA Free [NBP2-36468]

Western Blot: Histone H3 Antibody (1B1-B2) [NBP2-36468] - Analysis in Hela cells using siRNA to knockdown Pol II, controls are GAPDH and Histone H3. Theoretical molecular weight of Histone H3 is 15 kDa. Image from a verified customer review.
Histone H3 Antibody (1B1-B2) - BSA Free

Flow Cytometry: Histone H3 Antibody (1B1-B2) - BSA Free [NBP2-36468] -

Flow Cytometry: Histone H3 Antibody (1B1-B2) - BSA Free [NBP2-36468] - Analysis using Alexa Fluor (R) 647 conjugate of NBP2-36468. Jurkat cells stained with Alexa Fluor 647 conjugated Histone H3 antibody (red) or isotype control (blue). Flow cytometry image submitted by a verified customer review.

Applications for Histone H3 Antibody (1B1-B2) - BSA Free

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

1:200 -1:500

Immunohistochemistry

1:200-1:500

Immunohistochemistry-Paraffin

1:200-1:500

Single Cell Western

100 ug/ml

Western Blot

1:1000

Reviewed Applications

Read 1 review rated 5 using NBP2-36468 in the following applications:

Formulation, Preparation, and Storage

Purification

Protein G purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1.0 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: Histone H3

Chromatin is an arrangement of DNA and proteins which form chromosomes. Chromatin is formed from nucleosomes, which are comprised of a set of four histone proteins (H2A, H2B, H3, H4) wrapped with DNA. Chromatin is very dynamic, where post-translational modifications can regulate DNA to be copied, transcribed, or repaired.

Histones are nuclear proteins responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. Changes in chromatin structure play a large role in the regulation of transcription. The chromatin fibers are compacted through the interaction of a linker histone, H1, with the DNA between the nucleosomes to form higher order chromatin structures.

Common histone modifications include methylation of lysine and arginine, acetylation of lysine, phosphorylation of threonine and serine, and sumoylation, biotinylation, and ubiquitylation of lysine. Posttranslational modifications such as acetylation of core histones regulates gene expression, thus altering protein function and regulation (1). Histone H3 is primarily acetylated at lysines 9, 14, 18, and 23 and have a theoretical molecular weight of 15 kDa. Acetylation at lysine 9 and 14 appears to control histone deposition, chromatin assembly and active transcription. Methylation of arginine residues within histone H3 has also been linked to transcription regulation. Histone H3 has been linked to various types of cancer as a biomarker through the aberrant expression of histone deacetylase (HDAC) enzymes and changes to chromatins (2-4).

References

1. Zhang, Y. X., Akumuo, R. C., Espana, R. A., Yan, C. X., Gao, W. J., & Li, Y. C. (2018). The histone demethylase KDM6B in the medial prefrontal cortex epigenetically regulates cocaine reward memory. Neuropharmacology, 141, 113-125. doi:10.1016/j.neuropharm.2018.08.030

2. Nandakumar, V., Hansen, N., Glenn, H. L., Han, J. H., Helland, S., Hernandez, K,...Meldrum, D. R. (2016). Vorinostat differentially alters 3D nuclear structure of cancer and non-cancerous esophageal cells. Sci Rep, 6, 30593. doi:10.1038/srep30593

3. Zhou, M., Li, Y., Lin, S., Chen, Y., Qian, Y., Zhao, Z., & Fan, H. (2019). H3K9me3, H3K36me3, and H4K20me3 Expression Correlates with Patient Outcome in Esophageal Squamous Cell Carcinoma as Epigenetic Markers. Dig Dis Sci, 64(8), 2147-2157. doi:10.1007/s10620-019-05529-2

4. Li, Y., Guo, D., Sun, R., Chen, P., Qian, Q., & Fan, H. (2019). Methylation Patterns of Lys9 and Lys27 on Histone H3 Correlate with Patient Outcome in Gastric Cancer. Dig Dis Sci, 64(2), 439-446. doi:10.1007/s10620-018-5341-8

Alternate Names

H3F3A

Gene Symbol

H3C14

Additional Histone H3 Products

Product Documents for Histone H3 Antibody (1B1-B2) - BSA Free

Certificate of Analysis

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Product Specific Notices for Histone H3 Antibody (1B1-B2) - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Related Research Areas

Epigenetics
Interferons
Notch Pathway

Citations for Histone H3 Antibody (1B1-B2) - BSA Free

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Customer Images

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  • Name: Sara Smith
    Application: Western Blot
    Sample Tested: Whole cell lysate from HeLa cells
    Species: Human
    Verified Customer | Posted 12/09/2014
    Histone H3 WB in Hela cells
    Histone H3 Antibody (1B1-B2) - BSA Free NBP2-36468

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Protocols

View specific protocols for Histone H3 Antibody (1B1-B2) - BSA Free (NBP2-36468):

Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and wash the cells briefly in PBS. Add 10% formalin to the dish and fix at room temperature for 10 minutes.
2. Remove the formalin and wash the cells in PBS.
3. Permeablize the cells with 0.1% Triton X100 or other suitable detergent for 10 min.
4. Remove the permeablization buffer and wash three times for 10 minutes each in PBS. Be sure to not let the specimen dry out.
5. To block nonspecific antibody binding, incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
6. Add primary antibody at appropriate dilution and incubate overnight at 4C.
7. Remove primary antibody and replace with PBS. Wash three times for 10 minutes each.
8. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
9. Remove secondary antibody and replace with PBS. Wash three times for 10 minutes each.
10. Counter stain DNA with DAPi if required.

Immunohistochemistry-Paraffin Embedded Sections

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes (keep slides in the sodium citrate buffer all the time).

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in PBS for 5 minutes.
3. Block each section with 100-400 ul blocking solution (1% BSA in PBS) for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4 C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul HRP polymer conjugated secondary antibody. Incubate 30 minutes at room temperature.
7. Wash sections three times in wash buffer for 5 minutes each.
8. Add 100-400 ul DAB substrate to each section and monitor staining closely.
9. As soon as the sections develop, immerse slides in deionized water.
10. Counterstain sections in hematoxylin.
11. Wash sections in deionized water two times for 5 minutes each.
12. Dehydrate sections.
13. Mount coverslips.

Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10-25 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot TBS -0.05% Tween 20 (TBST).
5. Block the membrane in 5% Non-fat milk in TBST (blocking buffer) for at least 1 hour.
6. Wash the membrane in TBST three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate overnight at 4C with gentle rocking.
8. Wash the membrane in TBST three times for 10 minutes each.
9. Incubate the membrane in diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) for 1 hour at room temperature.
10. Wash the blot in TBST three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturers instructions.

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