Not all complexes are able to be captured in the “Catch-3” step of the affinity capture assay (Figure 5 ), and therefore analyzed by PAGE, because the 3′ end of the SOMAmer is sometimes involved in its structure or interaction with the target. Additional affinity capture examples for the subset of the CKD-related targets whose complexes can be captured on “Catch-3” beads are shown in Figure 5 .
50% plasma samples were prepared by diluting ethylene diamine tetraacetic acid (EDTA)-plasma 2X in SB18T with 2 µM Z-Block_2 (the modified nucleotide sequence (AC-BnBn)7-AC). The plasma spike samples were prepared by diluting 500 ng protein with the 50% plasma in SB17T (SB18T with 1 mM EDTA) with 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and ethylene glycol tetraacetic acid (EGTA). The plasma samples were prepared by diluting the 50% plasma in SB17T with AEBSF and EGTA. The buffer spike samples were prepared by diluting 500 ng protein in SB17T with AEBSF and EGTA. These samples were combined with 10 pmoles of SOMAmer to give final concentrations of 10% plasma, 2 mM AEBSF, 0.5 mM EGTA, and 100 nM SOMAmer. Complexes were formed by incubating at 37°C for 45 minutes. 50 µL of a 20% slurry of Streptavidin agarose beads (ThermoFisher Scientific) was added to each sample and shaken for 10 minutes at room temperature. The samples were added to a MultiScreen HV Plate to perform washes under vacuum filtration. Each sample was washed one time quickly with 200 µL of SB17T, one time for one minute with 200 µL of 100 µM biotin in SB17T with shaking, one time with 200 µL of SB17T for one minute with shaking, and one time with 200 µL of SB17T for nine minutes with shaking. Proteins in the sample were labeled with both biotin and a fluorophore by incubating each sample in 100 µL of 1 mM EZ Link NHS-PEO4-biotin (Pierce), 0.25 mM NHS-Alexa-647 (Invitrogen) in SB17T for five minutes with shaking. Each sample was washed one time with 200 µL of 20 mM glycine in SB17T and five times with 200 µL of SB17T, shaking each wash for one minute. The final wash was removed using centrifugation at 1000 relative centrifugal force (RCF) for 30 seconds. The beads were resuspended with 100 µL of SB17T. SOMAmers (complexed and free) were released from the beads by exposure under a BlackRay light source (UVP XX-Series Bench Lamps, 365 nm) for ten minutes with shaking. The samples were spun out of the plate by centrifugation at 1000 RCF for 30 seconds. 10 µL of each sample was removed and reserved as “Catch-1 eluate” for SDS-PAGE analysis. The remainder of the samples was captured through the biotinylated proteins by adding 20 µL of a 20% slurry of monomeric Avidin beads and shaking for ten minutes. The beads were transferred to a MultiScreen HV Plate and washed four times with 100 µL of SB17T for one minute with shaking. The final wash was removed using centrifugation at 1000 RCF for 30 seconds. Proteins were eluted from the beads by incubating each sample with 100 µL of 2 mM biotin in SB17T for five minutes with shaking. Each eluate was transferred to 0.4 mg MyOne Streptavidin beads with a bound biotinylated-primer complementary to the 3′ fixed region of the SOMAmer. The samples were incubated for five minutes with shaking to anneal the bead-bound fixed region to the SOMAmer complexes. Each sample was washed two times with 100 µL of 1XSB17T for one minute each with shaking and one time with 100 µL of 1XSB19T (5 mM HEPES, 100 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, 0.05% Tween-20, pH 7.5) for one minute with shaking, all by magnetic separation. The complexes were eluted by incubating with 45 µL of 20 mM NaOH for two minutes with shaking. 40 µL of each eluate was added to 10 µL of 80 mM HCl with 0.05% Tween-20 in a new plate. 10 µL of each sample was removed and reserved as “Catch-2 aptamer-bound eluate” for SDS-PAGE analysis. Gel samples were run on NuPAGE 4–12% Bis Tris Glycine gels (Invitrogen) under reducing and denaturing conditions according to the manufacturer's directions. Gels were imaged on an Alpha Innotech FluorChem Q scanner in the Cy5 channel to image the proteins.
50% plasma samples were prepared by diluting ethylene diamine tetraacetic acid (EDTA)-plasma 2X in SB18T with 2 µM Z-Block_2 (the modified nucleotide sequence (AC-BnBn)7-AC). The plasma spike samples were prepared by diluting 500 ng protein with the 50% plasma in SB17T (SB18T with 1 mM EDTA) with 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and ethylene glycol tetraacetic acid (EGTA). The plasma samples were prepared by diluting the 50% plasma in SB17T with AEBSF and EGTA. The buffer spike samples were prepared by diluting 500 ng protein in SB17T with AEBSF and EGTA. These samples were combined with 10 pmoles of SOMAmer to give final concentrations of 10% plasma, 2 mM AEBSF, 0.5 mM EGTA, and 100 nM SOMAmer. Complexes were formed by incubating at 37°C for 45 minutes. 50 µL of a 20% slurry of Streptavidin agarose beads (ThermoFisher Scientific) was added to each sample and shaken for 10 minutes at room temperature. The samples were added to a MultiScreen HV Plate to perform washes under vacuum filtration. Each sample was washed one time quickly with 200 µL of SB17T, one time for one minute with 200 µL of 100 µM biotin in SB17T with shaking, one time with 200 µL of SB17T for one minute with shaking, and one time with 200 µL of SB17T for nine minutes with shaking. Proteins in the sample were labeled with both biotin and a fluorophore by incubating each sample in 100 µL of 1 mM EZ Link NHS-PEO4-biotin (Pierce), 0.25 mM NHS-Alexa-647 (Invitrogen) in SB17T for five minutes with shaking. Each sample was washed one time with 200 µL of 20 mM glycine in SB17T and five times with 200 µL of SB17T, shaking each wash for one minute. The final wash was removed using centrifugation at 1000 relative centrifugal force (RCF) for 30 seconds. The beads were resuspended with 100 µL of SB17T. SOMAmers (complexed and free) were released from the beads by exposure under a BlackRay light source (UVP XX-Series Bench Lamps, 365 nm) for ten minutes with shaking. The samples were spun out of the plate by centrifugation at 1000 RCF for 30 seconds. 10 µL of each sample was removed and reserved as “Catch-1 eluate” for SDS-PAGE analysis. The remainder of the samples was captured through the biotinylated proteins by adding 20 µL of a 20% slurry of monomeric Avidin beads and shaking for ten minutes. The beads were transferred to a MultiScreen HV Plate and washed four times with 100 µL of SB17T for one minute with shaking. The final wash was removed using centrifugation at 1000 RCF for 30 seconds. Proteins were eluted from the beads by incubating each sample with 100 µL of 2 mM biotin in SB17T for five minutes with shaking. Each eluate was transferred to 0.4 mg MyOne Streptavidin beads with a bound biotinylated-primer complementary to the 3′ fixed region of the SOMAmer. The samples were incubated for five minutes with shaking to anneal the bead-bound fixed region to the SOMAmer complexes. Each sample was washed two times with 100 µL of 1XSB17T for one minute each with shaking and one time with 100 µL of 1XSB19T (5 mM HEPES, 100 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA, 0.05% Tween-20, pH 7.5) for one minute with shaking, all by magnetic separation. The complexes were eluted by incubating with 45 µL of 20 mM NaOH for two minutes with shaking. 40 µL of each eluate was added to 10 µL of 80 mM HCl with 0.05% Tween-20 in a new plate. 10 µL of each sample was removed and reserved as “Catch-2 aptamer-bound eluate” for SDS-PAGE analysis. Gel samples were run on NuPAGE 4–12% Bis Tris Glycine gels (Invitrogen) under reducing and denaturing conditions according to the manufacturer's directions. Gels were imaged on an Alpha Innotech FluorChem Q scanner in the Cy5 channel to image the proteins.
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