189
9. Prewash a strong anion-exchange spin column by applying
400 μL buffer A and spinning at 2000 × g for 5 min and discarding the flow-through.
10. Repeatedly add 400 μL of desalted ODN-protein conjugates
and centrifuge for 5 min at 2000 × g until the entire sample is
loaded on the anion-exchange spin column. Discard the
flow-through.
11. Remove non-conjugated protein by adding 400 μL buffer A and
centrifuging for 5 min at 2000 × g. Discard the flow- through.
Repeat this step ten times.
12. Elute the purified ODN-protein conjugate by adding 200 μL
buffer B and centrifuging for 5 min at 2000 × g. Repeat this
step ten times.
13. Analyze the elution fractions by 12% SDS-PAGE and combine
all fractions containing the purified ODN-protein conjugates
(Fig. 3).
14. Quantify the concentrations of the ODN-protein conjugates
by measuring the absorbance at λ = 260 nm and using extinction
coefficients of the oligonucleotides.
15. Aliquot the purified ODN-protein conjugates in 20 μL fractions
and flash-freeze in liquid nitrogen. Store the ODN- protein
conjugates at −80 °C.
Fig. 3 SDS-PAGE analysis of the conjugation and anion exchange purification of a maleimide-functionalized
oligonucleotide to a cysteine in (a) TEM1-β-lactamase and (b) BLIP on a 12% SDS-PAGE gel stained with
Coomassie blue. (rxn: reaction mixture, buffer A wash: first five wash fractions with buffer A (low ionic strength)
from the anion-exchange spin column, buffer B elution: first eight elution fractions with buffer B (high ionic
strength) from the anion-exchange spin column
DNA Sensors Based on β-Lactamase
9. Prewash a strong anion-exchange spin column by applying
400 μL buffer A and spinning at 2000 × g for 5 min and discarding the flow-through.
10. Repeatedly add 400 μL of desalted ODN-protein conjugates
and centrifuge for 5 min at 2000 × g until the entire sample is
loaded on the anion-exchange spin column. Discard the
flow-through.
11. Remove non-conjugated protein by adding 400 μL buffer A and
centrifuging for 5 min at 2000 × g. Discard the flow- through.
Repeat this step ten times.
12. Elute the purified ODN-protein conjugate by adding 200 μL
buffer B and centrifuging for 5 min at 2000 × g. Repeat this
step ten times.
13. Analyze the elution fractions by 12% SDS-PAGE and combine
all fractions containing the purified ODN-protein conjugates
(Fig. 3).
14. Quantify the concentrations of the ODN-protein conjugates
by measuring the absorbance at λ = 260 nm and using extinction
coefficients of the oligonucleotides.
15. Aliquot the purified ODN-protein conjugates in 20 μL fractions
and flash-freeze in liquid nitrogen. Store the ODN- protein
conjugates at −80 °C.
Fig. 3 SDS-PAGE analysis of the conjugation and anion exchange purification of a maleimide-functionalized
oligonucleotide to a cysteine in (a) TEM1-β-lactamase and (b) BLIP on a 12% SDS-PAGE gel stained with
Coomassie blue. (rxn: reaction mixture, buffer A wash: first five wash fractions with buffer A (low ionic strength)
from the anion-exchange spin column, buffer B elution: first eight elution fractions with buffer B (high ionic
strength) from the anion-exchange spin column
DNA Sensors Based on β-Lactamase