The effects of EDTA, dextran sulfate, and various detergents on DNA aptamer-conjugated magnetic bead pull-down of recombinant human ERK2 protein in buffer and human serum were investigated. Coomassie blue and silver-stained polyacrylamide gels revealed that a 192-base aptamer which was selected against a 19-amino acid peptide region of the human ERK2 protein in 25% trypsinized human plasma pulled down intact and fragmented ERK2 protein in phosphate buffered saline lacking calcium, but not in pure human serum. Addition of 2 mM EDTA enabled aptamer pulldown of ERK2 in serum. The identity of the recombinant ERK2 protein spiked into human serum samples and pulled down by aptamer-coated MBs was validated by mass spe... More
The effects of EDTA, dextran sulfate, and various detergents on DNA aptamer-conjugated magnetic bead pull-down of recombinant human ERK2 protein in buffer and human serum were investigated. Coomassie blue and silver-stained polyacrylamide gels revealed that a 192-base aptamer which was selected against a 19-amino acid peptide region of the human ERK2 protein in 25% trypsinized human plasma pulled down intact and fragmented ERK2 protein in phosphate buffered saline lacking calcium, but not in pure human serum. Addition of 2 mM EDTA enabled aptamer pulldown of ERK2 in serum. The identity of the recombinant ERK2 protein spiked into human serum samples and pulled down by aptamer-coated MBs was validated by mass spectrometry. Three unrelated aptamer sequences attached to MBs failed to pull down recombinant ERK2 in buffer. Electrophoretic banding pattern differences between various derivatized magnetic beads following exposure to human serum and acid elution were also examined in the presence and absence of dextran sulfate and various detergents to determine the best method for reducing nonspecific binding. Data suggest that additives can improve the binding affinity and specificity or purity of aptamer magnetic pull-down assays, but may also diminish target capture efficiency. Therefore, the specific effects of each additive on particular prototypical aptamer pull-down assays should be carefully studied and characterized by electrophoresis prior to use in more sophisticated mass spectral or other analytical techniques.
