Summary of solution phase PLA for detection of IL8 proteins in PLA buffer or in 10% or 50% chicken serum.

<p>R² is the correlation coefficient of the fitting curve from 100 pM to 0.16 pM. Background Ct equals the average Ct from quadruplicates of sample in reactions with no spiked IL8. Background CV% is the coefficient of variation calculated from the background Ct values for all quadruplicates.</p><p>Summary of solution phase PLA for detection of IL8 proteins in PLA buffer or in 10% or 50% chicken serum.</p

Solution phase PLA for detection of recombinant human IL8.

<p>(a) Detection of IL8 in PLA buffer with 60 pM pure anti-IL8 DNA conjugates (diamonds) and 60 pM unpurified anti-IL8 DNA conjugates (squares). (b) Detection of IL8 in PLA buffer (diamonds), 10% chicken serum (squares) and 50% chicken serum (triangles) using 60 pM pure anti-IL8 DNA conjugates. (c) Different amounts of unconjugated anti-IL8 (at a final concentration from 480 pM to 0 pM) were spiked in 60 pM of pure anti-IL8 conjugates. This series of mixes of probes were used in samples with either 10 pM or 0 pM IL8 in PLA buffer. (d) Different amounts of unconjugated oligonucleotides (Arm1 and Arm2 at a final concentration from 120 pM to 0 pM) were spiked in 60 pM of pure anti-IL8 conjugates. The probe mixes were employed in samples with 100 pM or 0 pM IL8 in PLA buffer. Error bars (a and b) indicate standard deviation from quadruplicate reactions and error bars (c and d) indicate standard deviation from duplicate reactions.</p

Gel electrophoresis of samples undergoing two-step purification.

<p>(a) Polyacrylamide gel of samples from purification of antibody-DNA conjugates and controls. Lane 1: PageRuler protein ladder (10–170 kDa, Thermo Scientific), Lane 2: 50 bp DNA ladder (Thermo Scientific), Lane 3: Arm1_long oligonucleotides (54 bp), Lane 4: Arm1 oligonucleotides (35 bp), Lane 5: anti-IL8 antibodies, Lane 6: conjugation reaction mixture of anti-IL8 and Arm1_long, Lane 7: conjugation reaction mixture of anti-IL8 and Arm1, Lane 8: eluate of captured sample of lane 6 after MlyI cleavage, Lane 9: anti-IL8 conjugates after two-step purification. The gel was stained with PlusOne DNA Silver Staining Kit. (b) Polyacrylamide gel of samples from purification of DARPin-DNA conjugates and controls. Lane 1: PageRuler protein ladder, Lane 2: 50 bp DNA ladder, Lane 3: S3block_long oligonucleotides (51 bp), Lane 4: S3block oligonucleotides (29 bp), Lane 5: G3-SNAP (34.5 kDa), Lane 6: conjugation reaction mixture of G3-SNAP and S3block_long, Lane 7: conjugation reaction mixture of G3-SNAP and S3block, Lane 8: eluate of sample of lane 6 after MlyI cleavage, Lane 9: G3-SNAP conjugates after the two-step purification. Relevant protein and DNA sizes are indicated. The gel was stained with Silver Stain for Mass Spectrometry kit.</p

Detection of HER2 proteins using immuno-RCA and <i>in</i><i>situ</i> PLA in fixed cells.

<p>(a) 5 nM pure G3-SNAP conjugates and 5 nM unpurified G3-SNAP conjugates were separately used in immune-RCA to detect HER2 proteins in HER2-positive (SK-OV-3) or -negative (BJhTERT) cells. Red dots represent RCA products arising from immuno-RCA reactions detected with fluorescence labeled complementary oligonucleotides. The numbers of RCA products per cell from those images are shown in bar charts (b). (c) Boxplots present the numbers of RCA products per cell when mixes of pure G3-SNAP conjugates (5 nM) and different amounts of unconjugated G3-SNAP (from 0 nM to 320 nM) were used to detect HER2 proteins via immuno-RCA in SK-OV-3 cells. (d) Boxplots reflect the number of RCA products per cell after <i>in</i><i>situ</i> PLA for detection of HER2 protein in SK-OV-3 cells, probed with pure probe conjugates (20 nM G3-SNAP conjugates and 20 nM 9.01-SNAP conjugates), mixed with different amounts of unconjugated DARPin binders (G3-SNAP and 9.03-SNAP at concentrations of 0 nM or 20 nM). The cell nuclei were counterstained with DAPI (blue).</p