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Applications: ChIP (1 - 5 µg/ChIP)
ELISA (1:5000)
WB (1:1000)
IF (1:500)
Assay Conditions: ChIP results obtained with the monoclonal antibody directed against H3K4un ChIP assays were performed using HeLa cells, the monoclonal antibody against H3K4un (Cat. No. 25258) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 10^5 cells. A titration of the antibody consisting of 1, 5 and 10 µg per ChIP experiment was analysed. IgG (5 µg/IP) was used as negative IP control. QPCR was performed with primers for the GAPDH promoter and for exon 2 of the myoglobin gene. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).Cross reactivity of the monoclonal antibody directed against H3K4un
To test the specificity an ELISA was performed using a serial dilution of the monoclonal antibody against H3K4un (Cat. No. 25258). The wells were coated with peptides containing the unmodified H3K4 region as well as the mono-, di- and trimethylated H3K4. Figure 2 shows a high specificity of the antibody for the unmodified peptide.Western blot analysis using the monoclonal antibody directed against H3K4un
Histone extracts (15 µg) from HeLa cells were analysed by Western blot using the monoclonal antibody against H3K4un (Cat. No. 25258) diluted 1:1,000 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.Immunofluorescence using the monoclonal antibody directed against H3K4un
HeLa cells were stained with the antibody against H3K4un (Cat. No. 25258) and with DAPI. Cells were fixed with 4% formaldehyde for 10 minutes and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with the H3K4un antibody (left) diluted 1:500 in blocking solution followed by an anti-mouse antibody conjugated to Alexa594. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.
Background: Histones are the main constituents of the protein part of chromosomes of eukaryotic cells. They are rich in the amino acids arginine and lysine and have been greatly conserved during evolution. Histones pack the DNA into tight masses of chromatin. Two core histones of each class H2A, H2B, H3 and H4 assemble and are wrapped by 146 base pairs of DNA to form one octameric nucleosome. Histone tails undergo numerous post-translational modifications, which either directly or indirectly alter chromatin structure to facilitate transcriptional activation or repression or other nuclear processes. In addition to the genetic code, combinations of the different histone modifications reveal the so-called "histone code". Histone methylation and demethylation is dynamically regulated by respectively histone methyl transferases and histone demethylases.
Concentration: 50 µg/50 µl
Cross Reactivity: Recognizes unmethylated H3K4 (HeK4me0). Does not significantly bind to methylated H3K4 (H3K4me1, H3K4me2 or H3K4me3).
Description: Monoclonal antibody raised in mouse against histone H3 using a KLH-conjugated synthetic peptide containing the unmodified lysine 4 (H3K4un)
Format: Aqueous buffer solution
Formulation: PBS containing 0.05% azide and 0.05% ProClin 300
Immunogen: synthetic peptide
Purification: Protein A purified
Storage Stability: Store at -80°C for up to 2 years. Centrifuge after first thaw to maximize product recovery. Aliquot to avoid repeated freeze/thaw cycles. Aliquots may be stored at -20°C for at least one month.
Uniprot: P68431
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
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