2.3 Protein-DNA interaction with DNA binding assay
Genomic DNA was extracted from HDF cells using HIMEDIA kit (Cat. No:
MB506-50PR) and restricted with a Rsa1 and Hinf1 restriction enzymes
purchased from New England Bio Labs (Catalog number R0167S and R0155S).
After digestion, telomeric repeats (TTAGGG)n were mixed with DNA coating
solution purchased from Thermo scientific (Catalog number 17250) and
coated to the microtiter plates. Coated microtiter plates were incubated
at RT for 1-2 hours with gentle shaking. Protein lysate was prepared
from HDF cells and incubated in telomeric DNA coated microtiter plates
for 3 hours at 4°C. Unbound proteins were washed with 1X PBS. Primary
antibodies (Lamin A (1:100) and trf2 (1:100)) in 1% BSA were prepared
and added to each well. Microtiter plates were incubated overnight at
4°C. After repetitive washing with 1X TBST buffer at RT, the plates were
incubated with secondary antibody (anti-rabbit IgG Alexa fluor 488
(1:1000) and anti-mouse IgG Alexa fluor 647 (1:1000)) respectively, in a
dark place for 2 hours at RT. Excitation and Emission readings of the
samples were taken using multi-plate reader at 488 and 647 nm,
respectively.