TY - JOUR
T1 - Regeneration of PEG slide for multiple rounds of single-molecule measurements
AU - Paul, Tapas
AU - Ha, Taekjip
AU - Myong, Sua
N1 - Funding Information:
We thank Dr. Patricia L. Opresko, Dr. Momčilo Gavrilov, Dr. Michael Banco, Dr. Adrian R. Ferré-D’Amaré, and Kevin Rhine for providing the POT1, Rep-X, PcrA, DHX36, and FUS proteins, respectively. We thank Dr. Laura Ganser for editing and Bersabel Wondimagegnhu for careful proofreading of the manuscript and Dr. Amarshi Mukherjee and Dr. Manindra Bera for helpful discussions.This work was supported by National Institute of Health General Medicine ( 1R0S1GM115631-01A1 to T.P. and S.M. and 1R01-CA 207342-01A1 and 1RF1 NS113636-01 to S.M.).
Publisher Copyright:
© 2021 Biophysical Society
PY - 2021/5/4
Y1 - 2021/5/4
N2 - Single-molecule fluorescence detection of protein and other biomolecules requires a polyethylene glycol (PEG)-passivated surface. Individual channels on a PEG-passivated slide are typically used only a few times, limiting the number of experiments per slide. Here, we report several strategies for regenerating PEG surfaces for multiple rounds of experiments. First, we show regeneration of DNA- or RNA-tethered surfaces by washing out the bound protein by 0.1% sodium dodecyl sulfate, which is significantly more effective than 6 M urea, 6 M GdmCl, or 100 μM proteinase K. Strikingly, 10 consecutive experiments in five different systems produced indistinguishable results both in molecule count and protein activity. Second, duplexed DNA unwound by helicase or denatured by 50 mM NaOH was reannealed with a complementary strand to regenerate the duplexed substrate with an exceptionally high recovery rate. Third, the biotin-PEG layer was regenerated by using 7 M NaOH to strip off NeutrAvidin, which can be reapplied for additional experiments. We demonstrate five cycles of regenerating antibody immobilized surface by which three different protein activity was measured. Altogether, our methods represent reliable and reproducible yet simple and rapid strategies that will enhance the efficiency of single-molecule experiments.
AB - Single-molecule fluorescence detection of protein and other biomolecules requires a polyethylene glycol (PEG)-passivated surface. Individual channels on a PEG-passivated slide are typically used only a few times, limiting the number of experiments per slide. Here, we report several strategies for regenerating PEG surfaces for multiple rounds of experiments. First, we show regeneration of DNA- or RNA-tethered surfaces by washing out the bound protein by 0.1% sodium dodecyl sulfate, which is significantly more effective than 6 M urea, 6 M GdmCl, or 100 μM proteinase K. Strikingly, 10 consecutive experiments in five different systems produced indistinguishable results both in molecule count and protein activity. Second, duplexed DNA unwound by helicase or denatured by 50 mM NaOH was reannealed with a complementary strand to regenerate the duplexed substrate with an exceptionally high recovery rate. Third, the biotin-PEG layer was regenerated by using 7 M NaOH to strip off NeutrAvidin, which can be reapplied for additional experiments. We demonstrate five cycles of regenerating antibody immobilized surface by which three different protein activity was measured. Altogether, our methods represent reliable and reproducible yet simple and rapid strategies that will enhance the efficiency of single-molecule experiments.
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U2 - 10.1016/j.bpj.2021.02.031
DO - 10.1016/j.bpj.2021.02.031
M3 - Article
C2 - 33675764
AN - SCOPUS:85102799101
SN - 0006-3495
VL - 120
SP - 1788
EP - 1799
JO - Biophysical journal
JF - Biophysical journal
IS - 9
ER -