Monoclonal antibody raised in mouse against histone H2B containing the phosphorylated Serine 14 (H2BS14p), using a KLH–conjugated synthetic peptide.

Determination of the antibody titer
To determine the titer of the antibody, an ELISA was performed using a serial dilution of the monoclonal antibody against H2BS14p (Cat. No. 25234).The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution (Figure 1), the titer of the antibody was estimated to be 1:2,000.

Cross reactivity tests using the monoclonal antibody against H2BS14p
To test the cross reactivity of the antibody against H2BS14p (Cat. No. 25234), a Dot Blot analysis was performed with peptides containing other histone phosphorylations and the unmodified H2BS14. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:2,000. Figure 2 shows a high specificity of the antibody for the modification of interest.

Western blot analysis using the antibody directed against H2BS14p
HeLa cells were treated with colcemid to block the cell cycle in metaphase and 15 μg of histone extracts of these cells were analysed by Western blot using the antibody against H2BS14p (cat. No. 25234) diluted 1:200 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.

50 µg/50 µl

PBS containing 0.05% azide and 0.05% ProClin 300

Human

Protein A purified

synthetic peptide

Aqueous buffer solution

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.

ELISA (1:100)DB (1:2000)
WB (1:200)

The optimal antibody concentration should be determined by the end–user.

Avoid freeze/thaw cycles

Antibodies

Antibody