Experiments were performed as described in (34 (link),36 (link)). Briefly, double-stranded DNA probes with 5′ Cy5 or FAM labels at one strand were prepared using an annealing buffer (20 mM Tris–HCl, 50 mM MgCl2, 50 mM KCl, pH 8.0). Purified protein samples and fluorescently labeled DNA were incubated for 1 h at RT in EMSA buffer. Mini gels were first pre-ran in 1× TG buffer (Biorad:1610734) at 200 V for 30 min. Then, samples were loaded, and gels were run for 30 min at 200 V at 4°C. Images are captured using an Amersham Typhoon 5 Biomolecular Imager and quantified using ImageQuantTL 7.0. DNA probes used are listed in (Supplement Table S1). Cooperativity factors for homodimers were calculated as described in (37 (link),38 (link)) and single tube dual color EMSAs with differently labelled DNA elements were performed as described in (39 (link)).
Typhoon 5 biomolecular imager
Manufactured by Cytiva
The Typhoon 5 Biomolecular Imager is a versatile imaging system designed for a wide range of applications in life science research. It is capable of detecting and quantifying various biomolecules, such as proteins, nucleic acids, and small molecules, using a range of fluorescent and luminescent detection methods.
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Lab products found in correlation
4 protocols using typhoon 5 biomolecular imager
1
Fluorescent EMSA for DNA-Protein Interactions
2
Northern Blot Analysis of RNA Transcripts
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Northern blots were performed using a protocol adapted from (31 (link)). Thermo Fisher Millennium RNA Markers and samples containing 15 μg RNA were run on 1% agarose gels containing formaldehyde (Thermo Fisher NorthernMax Denaturing Gel Buffer) and ethidium bromide. After washing the gel twice each with water and then 10× SSC, RNA was transferred to a GE Healthcare Hybond-N membrane via capillary transfer overnight. The transferred RNA was crosslinked to the membrane with UV light and imaged (ethidium signal) with a Bio-Rad ChemiDoc MP Imaging System in Flamingo mode. The membrane was incubated at 42°C overnight in Thermo Fisher ULTRAhyb-Oligo containing a 32P-radiolabeled single-stranded DNA oligo probe (see Supplementary Table S2 for sequences). The membrane was washed and then exposed to a storage phosphor screen overnight before acquiring images with a Amersham Typhoon 5 Biomolecular Imager. Complete details can be found in the Supplementary Methods.
3
High-Throughput Aminoacylation Screening
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Ten sequences were chosen from among
the top 20 peaks for experimental
testing (Supporting Table S1 ). The corresponding
DNA oligonucleotides were obtained from IDT (HPLC-purified) and RNA
was transcribed using T7 RNA polymerase. In addition, a control sample
of random pool sequences was used to determine baseline uncatalyzed
activity (k0A0, measured as a combined parameter). RNA was labeled using a 5′
EndTag Labeling Kit (Vector Laboratories) with Alexa 488 (Fisher),
and purified by phenol-chloroform extraction. Labeled RNA sequences
were then incubated (RNA concentration of 100 nM) with BYO for 90
min under conditions described as above for k-Seq.
Following desalting, samples were incubated with 2 μM streptavidin
for 15 min in 10 mM Tris (pH 7.0), then analyzed by native PAGE. Gels
were scanned and fluorescence was quantified with ImageQuant software
on an Amersham Typhoon 5 Biomolecular Imager. Bands corresponding
to the streptavidin complex and the free RNA band were quantified
to calculate the fraction of each sequence that had undergone aminoacylation.
Values determined by k-Seq were compared to gel shift
percentages (Supporting Figure S3A ) to
determine the average fraction loss l during streptavidin
bead pull-down. This value of l was used as a correction
factor when calculating catalytic enhancements using k-Seq data, as k0A0 was measured by gel-shift assay (also seeSupporting Table S1 ).
the top 20 peaks for experimental
testing (
DNA oligonucleotides were obtained from IDT (HPLC-purified) and RNA
was transcribed using T7 RNA polymerase. In addition, a control sample
of random pool sequences was used to determine baseline uncatalyzed
activity (k0A0, measured as a combined parameter). RNA was labeled using a 5′
EndTag Labeling Kit (Vector Laboratories) with Alexa 488 (Fisher),
and purified by phenol-chloroform extraction. Labeled RNA sequences
were then incubated (RNA concentration of 100 nM) with BYO for 90
min under conditions described as above for k-Seq.
Following desalting, samples were incubated with 2 μM streptavidin
for 15 min in 10 mM Tris (pH 7.0), then analyzed by native PAGE. Gels
were scanned and fluorescence was quantified with ImageQuant software
on an Amersham Typhoon 5 Biomolecular Imager. Bands corresponding
to the streptavidin complex and the free RNA band were quantified
to calculate the fraction of each sequence that had undergone aminoacylation.
Values determined by k-Seq were compared to gel shift
percentages (
determine the average fraction loss l during streptavidin
bead pull-down. This value of l was used as a correction
factor when calculating catalytic enhancements using k-Seq data, as k0A0 was measured by gel-shift assay (also see
4
Fluorescent Electrophoretic Mobility Shift Assay
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Double‐stranded DNA (dsDNA) probes with 5′ Cy5 or Cy3 dyes attached to the forward strand were prepared using an annealing buffer (20 mM Tris/HCl, 50 mM MgCl2, 50 mM KCl, pH 8.0). Protein samples and fluorescently labeled DNA are incubated for ∼2 h in EMSA buffer (10 mM Tris/HCl pH 8.0, 0.1 mg ml − 1 BSA, 50 µM ZnCl2, 100 mM KCl, 10% glycerol, 0.10% Igepal CA630, 2 mM βME). Gels were first preran using a 1 × TG buffer (Tris 0.25 mM, glycine 192 mM, pH 8.0) at 200 V for 30 min. Then, 10 µl samples were loaded and gels were run for 30 min at 200 V at 4 °C. Images are captured using an Amersham Typhoon 5 Biomolecular Imager and quantified using ImageQuantTL 7.0. DNA probes used are listed in supplementary table S4 , Supplementary Material online. Cooperativity factors were calculated as described in (Ng et al. 2012 (link)) for heterodimers and (Jerabek et al. 2017 (link); Wang et al. 2018 (link)) for homodimers.
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