Protein samples were denatured at 95 °C and resolved by 12% SDS-PAGE at 30 mA for 2 h. After transfer using Trans-Blot (Bio-Rad), nitrocellulose membranes were developed using the following primary antibodies: anti-His-tag (ab18184, dilution 1:1000), anti-DDDDK (ab49763, dilution 1:3000), anti-ALFA (FluoTag®-X2 anti-ALFA AlexaFluor 647, dilution 1:1000), anti-ZC3HAV1 (Proteintech 16820-1-AP, dilution 1:3000), anti-RPL4 (Proteintech 67028-1-Ig, dilution 1:10000), anti-RPS6 (Proteintech 14823-1-AP, dilution 1:500), anti-RYDEN (SHFL; Proteintech 27865-1-AP, dilution 1:1000). The following secondary antibodies were used: IRDye® 800CW Goat anti-rabbit (dilution 1:25000) and IRDye® 680RD Donkey anti-Mouse (dilution 1:15000; both LI-COR). Bands were visualized using an Odyssey Clx infrared imager system (LI-COR) or a Typhoon7000 (GE Healthcare).
Typhoon 7000
Manufactured by GE Healthcare
The Typhoon 7000 is a multipurpose imaging system designed for life science research. It utilizes fluorescence and phosphor imaging technologies to capture high-resolution images of a variety of sample types, including gels, blots, and microarrays.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant tl 7, by GE Healthcare (3 mentions)
Irdye 800cw goat anti rabbit, by LI COR (1 mentions)
Odyssey clx infrared imager system, by LI COR (1 mentions)
Irdye 680rd donkey anti mouse, by LI COR (1 mentions)
Trans blot, by Bio-Rad (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Perfecthyb, by Toyobo (1 mentions)
Bamh1, by Toyobo (1 mentions)
Nucleospin tissue, by Macherey-Nagel (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Prism software, by GraphPad (1 mentions)
α tubulin, by Merck Group (1 mentions)
L 14c leucine, by PerkinElmer (1 mentions)
Antibody for β actin, by Merck Group (1 mentions)
β tubulin, by Merck Group (1 mentions)
Prism 6, by GraphPad (1 mentions)
Ninhydrin spray reagent, by Merck Group (1 mentions)
15 protocols using typhoon 7000
1
Western Blot Analysis of Protein Samples
2
Measuring pRNA-3WJ Strand Displacement Kinetics
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The time constant (τ) of the pRNA-3WJ core strand was determined through electrophoresis mobility shift assays (EMSA). A single strand of the pRNA-3WJ fragments was radiolabeled at the 5′-terminus using γ-32P ATP (PerkinElmer) and denoted with an asterisk, as previously described (Binzel et al. 2014 (link)). The radiolabeled oligo was held at a constant concentration of 10 nM and incubated over varying time points ranging from 0 to 720 min with completed pRNA-3WJ structure at 100 nM concentrations. Samples were snap frozen on dry ice and then an electrophoresis 12% native polyacrylamide gel at 100 V for 2 h at 4°C in TBM running buffer was run. The gel was then imaged by transferring the radio signal to a phosphor screen for 12 h at −80°C and visualized using a Typhoon 7000 (GE). Band quantification was completed using ImageJ. The ratio of single strand to 3WJ was then calculated and plotted against time in OriginPro 8.5. The curve was fit using Equation 7 below:
where t is the time in seconds, and τ is the time constant at which 50% of the labeled 3WJ strand was in single-strand state and 50% had replaced unlabeled strand in the 3WJ complex (Novikova et al. 2010 (link)). These studies were completed as a way to compare the strand displacement between each of the pRNA-3WJ strands.
where t is the time in seconds, and τ is the time constant at which 50% of the labeled 3WJ strand was in single-strand state and 50% had replaced unlabeled strand in the 3WJ complex (Novikova et al. 2010 (link)). These studies were completed as a way to compare the strand displacement between each of the pRNA-3WJ strands.
3
MAIT-iPSC Genomic DNA Analysis
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Genomic DNA (3 μg) prepared from MAIT-iPSCs (L7−1 to -6, L11-1 to -4, L15-1 to -3, and L19-1 to -6) with NucleoSpin Tissue (MACHEREY-NAGEL) was digested with 40 U BamH1 (TOYOBO) at 37°C for 18 hr, separated by 0.9% agarose gel electrophoresis, and transferred onto Biodyne membranes (PALL Life Sciences). The membrane was pre-hybridized with PerfectHyb (TOYOBO) at 68°C for 60 min and hybridized with an α-32P-dCTP-labeled probe at 68°C for 18 hr. The probe was amplified with the primer set TRAV19 (forward) and TRAV19 (reverse) using genomic DNA from C57BL/6 as a template, and the resultant 350 bp PCR product was radiolabeled with the Random Primer DNA Labeling Kit version 2.0 (Takara) (Figure 1—figure supplement 1D ). The membrane was washed with 2× saline sodium citrate (SSC) and then 0.1× SSC buffer twice at 68°C, exposed to an imaging plate, and the signal was detected with a phosphoimager (Typhoon 7000, GE Healthcare).
4
Western Blot Analysis of MFG-E8 in RAW 264.7 Cells
5
Ago2 Binding Affinity Determination
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32P-labeled TTR RNA (100 pM) was incubated with 2-fold serial dilutions (125 nM to 3.8 pM) of RNA-free hAgo2 for 30 min. Samples were applied to a slot blot apparatus as described in (28 (link)). The protein–RNA complex was captured on a nitrocellulose membrane, and the free RNA was captured on a subsequent nylon membrane. For the competition assays, increasing concentrations of the competing RNA and 100 pM of the labeled RNA were mixed with 1 nM (final concentration) of RNA-free hAgo2 and incubated for 30 min. The protein-bound and unbound radiolabeled RNA were visualized by phosphorimaging (Typhoon 7000, GE Healthcare) and quantified using GeneTools software (Synoptics). The results of three experiments were analyzed using Prism software (GraphPad). Data are shown as means of bound RNA/(bound RNA+unbound RNA) plus and minus the standard deviation (SD). For the competition assays results are shown as mean of [1 – (bound RNA/(bound+unbound RNA))]± SD.
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32P-labeled TTR RNA (100 pM) was incubated with 2-fold serial dilutions (125 nM to 3.8 pM) of RNA-free hAgo2 for 30 min. Samples were applied to a slot blot apparatus as described in (28 (link)). The protein–RNA complex was captured on a nitrocellulose membrane, and the free RNA was captured on a subsequent nylon membrane. For the competition assays, increasing concentrations of the competing RNA and 100 pM of the labeled RNA were mixed with 1 nM (final concentration) of RNA-free hAgo2 and incubated for 30 min. The protein-bound and unbound radiolabeled RNA were visualized by phosphorimaging (Typhoon 7000, GE Healthcare) and quantified using GeneTools software (Synoptics). The results of three experiments were analyzed using Prism software (GraphPad). Data are shown as means of bound RNA/(bound RNA+unbound RNA) plus and minus the standard deviation (SD). For the competition assays results are shown as mean of [1 – (bound RNA/(bound+unbound RNA))]
6
Skeletal Muscle Surface Staining and Western Blot
7
Purification and Radiolabeling of 211At
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211At was produced by the 209Bi(α,2n)211At reaction, followed by separation and purification by a dry distillation method. 211At was dissolved in 100 μL of distilled water. The 211At-crude solution at a final concentration of 10 MBq/mL was mixed with AA as a reducing agent at a final concentration of 1.2 w/v% and sodium bicarbonate as a pH adjuster at a final concentration of 2.1 w/v% at pH 8.0 and allowed to stand for 1 h at ambient temperature. Separately, the 211At-crude solution was also mixed with the other reducing agents, namely cysteine, glutathione, sodium sulfite, or ferrous sulfate, at a final concentration of 1 w/v% under the same conditions. The resultant mixtures of 211At solutions were analyzed by thin-layer chromatography (TLC) using Typhoon 7000 (GE Healthcare). The reagents were purchased from Nacalai Tesque.
8
Quantifying Protein Synthesis via CFPS
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To quantify the amount of protein synthesized in iVAX reactions, two approaches were used. Fluorescence units of sfGFP were converted to concentrations using a previously reported standard curve (83 (link)). Yields of all other proteins were assessed via the addition of 10 μM l -14C-leucine (11.1 GBq mmol−1, PerkinElmer) to the CFPS mixture to yield trichloroacetic acid–precipitable radioactivity that was measured using a liquid scintillation counter as described previously (84 (link)). Soluble fractions were also run on an SDS-PAGE gel and exposed by autoradiography. Autoradiographs were imaged with Typhoon 7000 (GE Healthcare Life Sciences).
9
A3B Deaminase Activity Assay
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Purified A3Bctd-mycHis was diluted to 100 nM in reaction buffer containing 10 mM Hepes (pH 7.4), 5 mM EDTA, 50 mM NaCl, and 0.5 mM. MBP-BORF2 was serially diluted in the same buffer to concentrations of 200, 100, and 50 nM. Equal volumes of MBP-BORF2 and A3Bctd-mycHis were mixed together before adding oligonucleotide. The oligonucleotide with a 3′ FAM used as a substrate for A3Bctd was ordered from IDT and contains the preferred A3B trinucleotide sequence “TCA” (ATTATTATTATTCAAATGGATTTATTTATTTATTTATTTATTT-FAM). The oligonucleotide was diluted to 800 nM in reaction buffer. Equal volumes of substrate and protein complex were mixed together and incubated for 1 hour at 37°C (total reaction volume, 10 μl). This was followed by addition of UDG (Uracil-DNA Glycosylase) (NEB, M0280L) for 10 min to remove the uracil base resulting from A3B deaminase activity. The reaction mix was then incubated with 1 M NaOH for 10 min at 98°C to break the phosphodiester backbone at the abasic site. Loading dye (11 μl) was added to each reaction, and reaction products were separated on a 15% tris-borate EDTA–urea acrylamide gel. The gel was imaged using Typhoon 7000 (GE Healthcare).
10
Quantitative Dot Blot Analysis
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