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Supporting Information
Selective detection of protein homologues in serum using an OmpG nanopore
Monifa A. Fahie‡, Bib Yang†, Martin Mullis†§, Matthew A. Holden† and Min Chen‡ †*
‡Molecular and Cellular Biology Program and †Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts 01003 * [email protected]
§
Current address: Molecular and Computational Biology program, University of Southern California, Los Angeles, CA 90089-0371
TABLE OF CONTENTS
Single Channel analysis S-2
Figure S1 S-2 Figure S2 S-3 Figure S3 S-3 Figure S4 S-4 Figure S5 S-5 Figure S6 S-6 Table S1 S-7 Table S2 S-8
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Single Channel analysis
OmpG gating was analyzed in Clampfit 10.3 using single channel search from a 5 s section of a trace. The extracted dwell times of the open and closed state were used to calculate the open probability, the inter-event duration, event duration, as well as the gating frequency. To determine the open probability, the sum of the open dwell times (τon) were divided by the total time analyzed. To determine the inter-event duration, the open dwell times (τon) were plotted in a histogram and fit with a single exponential function. The event duration was similarly calculated, but the closed dwell times (τoff) were fit instead. For gating frequency, the trace was first filtered with a low pass Gaussian filter with a 1 kHz -3dB cut off. The total count of gating events was divided by the total time. To determine the open pore conductance, an all-events histogram of the trace was fit with a Gaussian function.
Figure S1: SDS-PAGE analysis of refolding and labeling efficiency of OmpG-biotin. After
three days refolding at 37°C, samples of the OmpG-biotin protein was heated to 95°C for 15 minutes in Laemelli buffer or loaded without boiling to the 15% gel. To determine the labeling efficiency, the sample was mixed with streptavidin in a 1:1 molar ratio for 1 minute and then mixed with Laemelli buffer without boiling. Labeled OmpG forms a SDS-resistant complex with streptavidin which migrate slower in the gel. The intensity of the protein bands were analyzed by gel imager to calculate the labeling efficiency which was about 85%.
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Figure S2: Single channel current trace of OmpG D224C. SB58C (2 nM) was added to
unlabeled OmpG D224C and the ionic current monitored. There was no change in OmpG ionic current behavior. Buffer used was 10 mM sodium phosphate pH 6, 300 mM KCl and the ionic current recorded at both -50 and +50 mV. OmpG was recorded with a 2 kHz Bessel filter at a sampling rate of 100 µs.
Figure S3: Gating characteristics of the OmpG ionic current. Three of the five gating
parameters are defined in the trace. Open probability is the ratio of the open and closed state while gating frequency is the number of events per second. This trace is the SB binding states in the absence of serum at +50 mV. Buffer used was 10 mM sodium phosphate pH 6, 300 mM KCl. SB binding was recorded with a 2 kHz Bessel filter at a sampling rate of 100 µs.
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Figure S4: Detection of SB58C by OmpG-biotin nanopore. At both -50 and +50 mV,
binding state C was the predominant state. Binding state B was completely abolished, while binding state A was periodically seen at +50 mV. Buffer used was 10 mM Tris-HCl pH 8, 1.0 M KCl. Traces were recorded with a 2 kHz Bessel filter at a sampling rate of 100 µs.
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Figure S5: Simultaneous detection of two antibodies in serum. A continuous trace (170 s)
of SB (1 nM, blue) and BT (5 nM, red) binding to OmpG-biotin in the absence of 10% (v/v) serum. Buffer used was 10 mM sodium phosphate pH 6, 300 mM KCl and the ionic current recorded at -50 mV. SB and BT were recorded with a 2 kHz Bessel filter at a sampling rate of 100 µs.
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Figure S6: Effect of FBS on the kinetics of antibody binding. Histograms of the interevent duration (τon) and dwell time (τoff) of mAb binding events were fitted with single exponential decay function to derive the average time constants. Tau on values for BT were calculated from 10 nM. Buffer used was 10 mM sodium phosphate pH 6, 300 mM KCl and the ionic current was recorded at both -50 mV. Averages and standard deviations were taken from three independent pores.
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Table S1: The relative change in open probability, gating frequency and open current of
OmpG-biotin during type A, B and C binding of SB58C
Loop Voltage (mV) SB58C binding state Relative Open Probability Relative Gating Frequency Relative Open pore current -50 No bind 1.00 ± 0.04 1.00 ± 0.22 1.00 ± 0.05 Type A 1.31 ± 0.06 N.D. 0.62 ± 0.02 Type B 0.16 ± 0.05 0.46 ± 0.06 0.07 ± 0.02 Type C 0.79 ± 0.07 1.1 ± 0.04 0.94 ± 0.04 +50 No bind 1.00 ± 0.02 1.00 ± 0.09 1.00 ± 0.03 Type A 1.22 ± 0.03 N.D. 0.54 ± 0.03 Type B 0.19 ± 0.05 0.55 ± 0.10 0.04 ± 0.01 Type C 0.83 ± 0.06 1.38 ± 0.08 0.92 ± 0.05 *The errors indicate standard deviations from at least three independent pores.
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Table S2: The gating characteristics of the unbound state, the BT and SB bound states in the
absence and presence of 10% (v/v) serum.
Open probability Gating Frequency (s-1) Interevent duration (ms) Event duration (ms) Open pore conductance (pA) No bind - FBS 0.78 ± 0.03 68 ± 15 6.37 ± 1.88 2.23 ± 0.50 -29.3 ± 1.4 No bind + FBS 0.73 ± 0.06 121 ± 13 0.96 ± 0.31 1.35 ± 0.32 -30.1 ± 2.3 BTN.4 - FBS 0.52 ± 0.08 113 ± 4 0.97 ± 0.21 1.76 ± 0.34 -27.7 ± 1.3 BTN.4 + FBS 0.48 ± 0.16 146 ± 24 0.56 ± 0.19 1.46 ± 0.34 -28.7 ± 2.1 SB58C Type A - FBS 1.00 ± 0.00 0 ± 0 N.D. N.D. -18 ± 0.6 SB58C Type A + FBS 1.00 ± 0.00 0 ± 0 N.D. N.D. -20.2 ± 1.0 SB58C Type B - FBS 0.13 ± 0.04 32 ± 4 0.70 ± 0.04 3.92 ± 0.23 -2.1 ± 0.5 SB58C Type B + FBS 0.12 ± 0.04 20 ± 5 0.44 ± 0.03 2.16 ± 0.08 -3.8 ± 0.9 SB58C Type C - FBS 0.60 ± 0.06 77 ± 3 1.41 ± 0.07 0.86 ± 0.3 -27.5 ± 1.0 SB58C Type C + FBS 0.60 ± 0.11 121 ± 13 0.39 ± 0.10 0.74 ± 0.21 -29.9 ± 1.2 *The error bars indicate standard deviations from at least three independent pores.