Multiplexed analyses of test samples
A technology for detecting samples and components, which is applied in the determination/inspection of microorganisms, organic chemistry, biochemical equipment and methods, etc.
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Embodiment 1
[0207] Example 1. Aptamers and Primer Constructs
[0208] Aptamer and biotinylated primer constructs were generated with different 5' terminal functional groups, the differences are shown in Figure 6 . The aptamer contains a Cy3 fluorescent dye (Cy3 phosphoramidite, from GlenResearch (-[3-(4-methoxytrityloxy)propyl]-1′-[3-[( 2-cyanoethyl)-(N,N-diisopropyl)phosphoramidite]propyl]-3,3,3′,3′-tetramethylindocarbocyanine chloride)), primer Contains two biotin residues ((AB) 2 ), a (T) 8 Linker and a photocleavable moiety (PC linker as phosphoramidite (-(4,4'-dimethoxytrityl)-1-(2-nitrophenyl)-prop-1-yl-[ (2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite was obtained from Glen Research). For Figure 6 The method, the aptamer contains a photoreactive crosslinking group referred to herein as ANA (4-azido-2-nitroaniline), a photocleavable moiety (PC linker) and a Cy3 dye at the 5' end, and a primer containing Two biotin residues and a (T) 8 connector.
Embodiment 2
[0209] Example 2 . Affinity Binding Method (Double Capture (2Catch) Method)
[0210] a) Buffer
[0211] 30 μL of Cy3-aptamer mix (2 nM of each aptamer) was combined with 30 L of (AB)2-T8-PC-primer mix (6 nM of each primer) in SB17T and incubated at 95 °C for 4 min, at 37 °C 13 minutes. In another reaction, prepare 60 μL of target protein mix (2X concentration in SB17T). 55 μL of the target protein mix was combined with 55 μL of the aptamer / primer mix in a 96-well plate (Omni-TubePlate, Abgene #AB0407) and incubated at 37°C for 15 minutes to achieve binding equilibrium. All subsequent steps were performed at room temperature unless otherwise stated.
[0212] b) plasma, serum or whole blood
[0213] 30 μL of Cy3-aptamer mix (2 nM of each aptamer) was combined with 30 μL of (AB)2-T8-PC-primer mix (6 nM of each primer) in SB17T and incubated at 95 °C for 4 min, at 37 °C Keep warm for 13 minutes. In another reaction, in a Z-blocked competitor oligonucleotide (5′-(ACZZ) 7...
Embodiment 3
[0229] Example 3. Photocrosslinking Assay Protocol
[0230] All steps of this protocol were performed under conditions of minimal light exposure to prevent photoactivation of photoaptamers.
[0231] a) Protein binding
[0232] 30 μL of ANA-PC-Cy3-aptamer mix (2 nM of each aptamer) and 30 μL of (AB)2-T8-primer mix (6 nM of each aptamer) in SB17T buffer (40 mM HEPES, pH 7.5 , 120mM NaCl, 5mM KCl, 5mM MgCl 2 , 1 mM EDTA, 0.05% TWEEN-20) and incubated at 95°C for 4 minutes and at 37°C for 13 minutes. In another reaction, prepare 60 µL of protein mixture at 2X concentration. 55 μL of the target protein mix was combined with 55 μL of the aptamer / primer mix in a 96-well plate (Omni-Tube Plate, Abgene #AB0407) and incubated at 37°C for 15 minutes to achieve binding equilibrium. All subsequent steps were performed at room temperature unless otherwise stated.
[0233] b) Kinetic attack and photocrosslinking
[0234] 100 μL of the equilibrated sample was added to 1400 μL of SB17T...
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