WB was performed on whole cell (25 ug, lane 1) and histone extracts (15 ug, lane 2 ) from HeLa cells, and on 1 ug of recombinant histone H2A, H2B, H3 and H4 (lane 3, 4, 5 and 6, respectively) using the antibody against H2Apan. The antibody was diluted 1:2,000 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.
Determination of the titer To determine the titer of the antibody, an ELISA was performed using a serial dilution of the antibody against H2Apan in antigen coated wells. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the antibody was estimated to be 1:32, 500.
ChIP assays using HeLa cells (sheared chromatin from 1 million cells). A titration of the antibody consisting of 1, 2, 5, and 10 ug per ChIP experiment was analysed. IgG (5 ug/IP) was used as negative control. qPCR primers were for the GAPDH and EIF4A2 promotersas negative controls and for the inactive MYOD1 gene and the Sat2 satellite repeatas positive controls. Image shows the recovery, expressed as a % of input (the relative amount of IP'd DNA compared to input DNA after qPCR).
Supplier Page from OriGene Technologies for H2AW Rabbit Polyclonal Antibody