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Applications: ChIP (5 - 10 µg)
ELISA (1:100)
DB (1:20,000 )
WB (1:1000)
IF (1:500)
Assay Conditions: ChIP results obtained with the antibody directed against H3K36me1
ChIP assays were performed using human U2OS cells, the antibody against H3K36me1 (Cat. # 25246) and optimized PCR primer pairs for qPCR. ChIP was performed using sheared chromatin from 1.5 million cells and stringent washing conditions. A titration consisting of 1, 2, 5 and 10 µg of antibody per ChIP experiment was analyzed. IgG (1 µg/IP) was used as a negative IP control. Quantitative PCR was performed with primers for the promoter of the constitutively expressed GAPDH gene and for the inactive gene MYOD. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody directed against H3K36me1 (Cat. # 25246), crude serum and flow-through. 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 1:46,000.Cross reactivity tests using the antibody directed against H3K36me1
A Dot Blot analysis was performed to test the cross reactivity of the antibody against H3K36me1 (Cat. # 25246) with peptides containing other modifications of histone H3 or the unmodified H3K36. One hundred to 0.2 pmol of the peptide containing the respective histone modification were spotted on a membrane. 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.Western blot analysis using the antibody directed against H3K36me1
Histone extracts (15 µg) from HeLa cells were analyzed by Western blot using the antibody against H3K36me1 (Cat. # 25246) 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 antibody directed against H3K36me1
Human osteosarcoma (U2OS) cells were stained with the antibody against H3K36me1 (Cat. # 25246) and with DAPI. Cells were fixed with 4% formaldehyde for 20 minutes and blocked with PBS/TX-100 containing 5% normal goat serum. Figure 5A: cells were immunofluorescently labeled with the H3K36me1 antibody (left) diluted 1:500 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa568 or with DAPI (right), which specifically labels DNA. Figure 5B, C, D and E: staining of the cells with the H3K36me1 antibody after incubation of the antibody with 2 ng/µl blocking peptide containing the unmodified and the mono-, di- and trimethylated H3K36, respectively.
Concentration: 50 µg/54 µl
Description: Polyclonal antibody raised in rabbit against histone H3 containing the monomethylated lysine 36 (H3K36me1), using a KLH-conjugated synthetic peptide
Format: Aqueous buffer solution
Formulation: PBS containing 0.05% azide and 0.05% ProClin 300.
Immunogen: synthetic peptide
Purification: Affinity 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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