Polyclonal antibody raised in rabbit against the region of histone H2A.X containing the phosphorylated serine 139 (H2A.XS139p), 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 antibody directed against H2A.XS139p (Cat. No. 25227) in antigen coated wells. 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 1:170,000.

Cross reactivity tests using the antibody directed against H2A.XS139p
To test the cross reactivity of the antibody against H2A.XS139p (Cat. No. 25227), a Dot Blot analysis was performed with peptides containing other histone phosphorylations and the unmodified H2A.X. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:10,000. Figure 2 shows a high specificity of the antibody for the modification of interest.

Western blot analysis using the antibody directed against H2A.XS139p
Western blot was performed on histone extracts (15 μg) from untreated U2OS cells (lane 1) or from U2OS cells treated with camptothecin (lane 2), and on 1 μg of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the antibody against H2A.XS139p (Cat. No. 25227). The antibody was diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The marker (in kDa) is shown on the left.

50 µg/ 19 µl

PBS containing 0.05% azide and 0.05% ProClin 300

Human

Affinity 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 (1:10,000)
DB (1:10,000)
WB (1:1000)

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

Avoid freeze/thaw cycles

Antibodies

Antibody