M9-9 Aptamer Details

y, flat-bottom MaxiSorb microtiter plates, Nalge Nunc International (Naperville, IL) were coated with 90.9nM mCTLA-4/Fc in 0.05M carbonate-bicarbonate buffer pH 9.6 and incubated for 3 hours at room temperature with agitation. Plates were washed 5 times with HEPES wash buffer: 20mM HEPES pH 7.4, 0.15M NaCl, 2mM CaCl2, then blocked by addition of Super Block, Pierce (Rockford, IL) 3 times followed by washing 5 times. Serial dilutions of biotinylated Del 60 aptamer were done in HEPES binding buffer (HEPES wash buffer + 0.01% BSA) then incubated in the wells for 15 minutes at 37o C. Following one wash with HEPES buffer, streptavidin-AP (Jackson ImmunoResearch, West Grove, PA) was prepared in HEPES buffer then added to the wells and incubated for 15 minutes at room temperature. Excess streptavidin-AP was removed by washing 3 times with HEPES buffer then alkaline phosphatase substrate (Sigma, St. Louis, MO) was added.

If the oligo is a known aptamer sequence: For binding studies, perform a refolding protocol to ensure proper function (i.e. binding to antigen or target). Refer to the aptamer reference source for the appropriate refolding parameters and binding conditions. Note: it is unknown whether aptamer functions properly without refolding.