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Applications: ChIP (1 - 5 µl/ChIP)
ELISA (1:100 - 1:1000)
DB (1:1000)
WB (1:250)
Assay Conditions: Determination of the titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody directed against H3pan (Cat. # 25235). By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:900,000.Cross reactivity test using the antibody directed against H3pan
A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3pan (Cat. # 25235) with the peptides used for immunization of the rabbit and other peptides containing different modifications or unmodified sequences of histone H3. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:1,000. Figure 2 shows a high specificity of the antibody for one of the unmodified peptides.Western blot analysis using the antibody directed against H3pan
Histone extracts of HeLa cells (15 µg) were analyzed by Western blot using the antibody against H3pan (Cat. # 25235) diluted 1:250 in TBS-Tween containing 5% skimmed milk (lane 1). Lane 2 shows a coomassie blue staining of the gel. The position of the protein of interest is indicated on the left; the marker (in kDa) is shown on the right.ChIP results obtained with the antibody directed against H3pan
ChIP assays were performed using human HeLa cells, the antibody against H3pan (Cat. # 25235) and optimized PCR primer sets for qPCR. ChIP was performed on sheared chromatin from 10,000 cells per IP. A titration of the antibody consisting of 1, 5, and 10 µl per ChIP experiment was analyzed. IgG (5 µg/IP) was used as negative IP control. QPCR was performed with primers for the GAPDH promoter, MYOD, and the heterochromatin marker Sat2. Figure 4 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).
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. Histones play a central role in the regulation of transcription, DNA repair, DNA replication and chromosomal stability. These different functions are established via a complex set of post-translational modifications which either directly or indirectly alter chromatin structure and DNA accessibility to facilitate transcriptional activation or repression or other nuclear processes.
Concentration: 100 µl
Description: Polyclonal antibody raised in rabbit against two KLH-conjugated synthetic peptides containing an unmodified sequence from the central part and from the C-terminus of histone H3, respectively.
Format: Aqueous solution
Formulation: Whole antiserum from rabbit containing 0.05% azide
Immunogen: synthetic peptide
Purification: Whole serum
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.
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
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