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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [2]
- ELISA [1]
- Chromatin Immunoprecipitation [1]
- Other assay [1]
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- Product number
- NBP2-59247 - Provider product page
- Provider
- Novus Biologicals
- Product name
- Rabbit Polyclonal Histone H4 Antibody
- Antibody type
- Polyclonal
- Description
- Whole Antiserum.
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 ul
- Storage
- Store at -20C. Avoid freeze-thaw cycles.
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Supportive validation
- Submitted by
- Novus Biologicals (provider)
- Main image
- Experimental details
- Western Blot: Histone H4 [Methyl Lys20] Antibody [NBP2-59247] - Histone extracts of HeLa cells (15 ug) were analyzed using the antibody against H4K20me1 diluted 1:750 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (in kDa) is shown on the left.
- Submitted by
- Novus Biologicals (provider)
- Main image
- Experimental details
- Western Blot: Histone H4 [Methyl Lys20] Antibody [NBP2-59247] - Histone extracts of HeLa cells (15 ug) were analysed by Western blot using the antibody against H4K20me1 diluted 1:750 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right; the marker (kDa) is shown on the left.
Supportive validation
- Submitted by
- Novus Biologicals (provider)
- Main image
- Experimental details
- ELISA: Histone H4 [Methyl Lys20] Antibody [NBP2-59247] - To determine the titer, an ELISA was performed using a serial dilution of the antibody against H4K20me1. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:8,000.
Supportive validation
- Submitted by
- Novus Biologicals (provider)
- Main image
- Experimental details
- Chromatin Immunoprecipitation: Histone H4 [Methyl Lys20] Antibody [NBP2-59247] - ChIP assays were performed using human osteosarcoma (U2OS) cells, the antibody against H4K20me1 and optimized PCR primer sets for qPCR. Chromatin was sheared. ChIP was performed using sheared chromatin from 1.6 million cells. A titration of the antibody consisting of 2, 5, 10 and 15 ul per ChIP experiment was analysed. IgG (5 ug/IP) was used as negative IP control. Quantitative PCR was performed with primers for the GAPDH promoter and for the coding region of the myogenic differentiation gene (MYOD), a gene that is inactive at normal conditions. Figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
Supportive validation
- Submitted by
- Novus Biologicals (provider)
- Main image
- Experimental details
- Dot Blot: Histone H4 [Methyl Lys20] Antibody [NBP2-59247] - To check the specificity of the antibody against H4K20me1, a Dot Blot was performed with peptides containing other modifications of histone H4 or the unmodified sequence. Other histone modifications include mono- and dimethylation of the same lysine and acetylation of the nearby lysine 16. To determine the cross reactivity, 0.2 to 100 pmol of peptides were spotted on a membrane. Three different peptides for H4K16ac were used. The antibody was used at a dilution of 1:20,000. Figure 3 shows a high specificity of the antibody for the modification of interest.
