Peptide Dot Blot

The assay was performed using specific antibodies and dilutions of several peptides which were plotted onto nitrocellulose membrane. The specificity of antibodies we produced against CenH3s was tested on specific and nonspecific peptides. Synthetized 14–18mer peptides were dissolved in PBS to 1 mg/ml and then further diluted to concentration of 1, 0.1, and 0.01 µg/µl. The series of peptide dilutions in form of 2 µl spots were applied to the nitrocellulose membrane; the membrane was dried for 30 min and blocked in 5% BSA in TBST buffer for 1 h with mild shaking at RT. Affinity purified monospecific IgG fractions of anti-CenH3s were used as primary antibodies, diluted 1:500 in blocking buffer. For βCenH3-1, βCenH3-2, βγCenH3 antibody testing, two rabbits or three guinea pigs antibodies were pooled together when testing rabbit of guinea pig antibodies, respectively. After 1 h incubation at RT, membranes were washed 3 × 5 min with TBST and incubated with secondary antibody (HRP-conjugated antirabbit; Cell Signaling Technology, 7074) or antiguinea pig (Invitrogen, A18769) antibody diluted 1:1,000 in blocking buffer for 1 h at RT followed by 3 × 5 min washes with TBST. The final detection was carried out as described for the Western blot.