At 48h after transfection, cells were rinsed twice with PBS and then harvested in PBS containing protease inhibitors (Complete tablet from Roche). The cells were lysed in PBS, 1% Igepal and protease inhibitors for 30min on ice. The detergent lysates were then clarified by centrifugation (14,000×g, 30min, 4°C). Proteins were separated by SDS-PAGE on 3-8% Tris-Acetate or 4-12% Bis-Tris gels and then transferred to polyvinylidene fluoride membranes. After blocking in TBS buffer (10mM Tris, pH 7.4, 500mM NaCl. 0.5% Igepal, 10% goat serum and 3% BSA), the membranes were incubated with primary antibody overnight. The protein-Ab complexes were then labeled with a horseradish peroxidase-conjugated secondary Ab (1:3000, Sigma-Aldrich) for 1h at room temperature and detected using the enhanced ECL Plus reagent (GE Healthcare) visulalized with a Typhoon 9410 scanner (GE Healthcare). Quantification of immunoblot bands was performed with ImageQuant software (GE Healthcare). The following Abs were used: rabbit anti-CaV2.2 (1:500)53 (link), rabbit anti-CaVβ1b (1:500)22 (link) and mouse anti-CaVα2δ-1 (1:3000, D219, Sigma).
Typhoon 9410 scanner
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The Typhoon 9410 scanner is a high-performance imaging system designed for life science applications. It features a versatile scanning platform that can be used for a variety of detection methods, including fluorescence and chemiluminescence.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant software, by GE Healthcare (5 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Complete tablet, by Roche (3 mentions)
Horseradish peroxidase conjugated secondary ab, by Merck Group (3 mentions)
Imagequant tl software, by GE Healthcare (3 mentions)
Dneasy blood and tissue kit, by Qiagen (3 mentions)
Sequagel ureagel system, by National Diagnostics (3 mentions)
Ecl plus reagent, by GE Healthcare (2 mentions)
Imagequant 5, by GE Healthcare (2 mentions)
Hinfi, by New England Biolabs (2 mentions)
Chef dr 2 pulse field gel electrophoresis system, by Bio-Rad (2 mentions)
Phosphorimager screen, by GE Healthcare (2 mentions)
Zeta probe, by Bio-Rad (1 mentions)
Ipg strip, by GE Healthcare (1 mentions)
Ettan ipgphor 2, by GE Healthcare (1 mentions)
Colloidal coomassie, by Bio-Rad (1 mentions)
Rehydration tray, by GE Healthcare (1 mentions)
Bind silane, by GE Healthcare (1 mentions)
Se600 system, by Hoefer (1 mentions)
Nylon membrane, by GE Healthcare (1 mentions)
52 protocols using typhoon 9410 scanner
1
Western Blot Analysis of Cav Subunits
2
Quantitative Proteomic Analysis Using 2D-DIGE
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The
protein extracted (50 μg) from each sample underwent Cy3 labeling
or Cy5 labeling. Also, a mixture of an equal amount of all samples
was pooled, labeled with Cy2, and used as an internal standard as
described previously.58 (link)−61 (link) During labeling, dye switching was applied to avoid a dye-specific
bias (Table S1 ). First- and second-dimension
analytical gel electrophoresis was carried out as described previously.58 (link)−61 (link) Furthermore, a Typhoon 9410 scanner (GE Healthcare) was used for
imaging the 2D-DIGE gels using excitation/emission wavelengths specific
for Cy2 (488/520 nm), Cy3 (532/580 nm), and Cy5 (633/670 nm).
The Coomassie Blue-stained gel was washed and digested from a preparatory
gel according to procedures reported previously.58 (link)−60 (link) Spotting was
carried out onto a MALDI target (384 MTP Anchorchip; 800 pm Anchorchip;
Bruker Daltonics, Bremen, Germany) from a mixture of tryptic peptides
(1 pL) derived from each protein. MALDI-MS(/MS) spectra were recorded
using an UltrafleXtreme TOF mass spectrometer with a reflector voltage
and a detector voltage of 21 and 17 kV, respectively, as reported.62 (link),63 (link) The Mascot search algorithm v2.0.04 (updated on December 9, 2019;
Matrix Science, London, UK) was used for searching peptide masses.
The identified proteins were assessed for a Mascot score > 56 and p < 0.05.
protein extracted (50 μg) from each sample underwent Cy3 labeling
or Cy5 labeling. Also, a mixture of an equal amount of all samples
was pooled, labeled with Cy2, and used as an internal standard as
described previously.58 (link)−61 (link) During labeling, dye switching was applied to avoid a dye-specific
bias (
analytical gel electrophoresis was carried out as described previously.58 (link)−61 (link) Furthermore, a Typhoon 9410 scanner (GE Healthcare) was used for
imaging the 2D-DIGE gels using excitation/emission wavelengths specific
for Cy2 (488/520 nm), Cy3 (532/580 nm), and Cy5 (633/670 nm).
The Coomassie Blue-stained gel was washed and digested from a preparatory
gel according to procedures reported previously.58 (link)−60 (link) Spotting was
carried out onto a MALDI target (384 MTP Anchorchip; 800 pm Anchorchip;
Bruker Daltonics, Bremen, Germany) from a mixture of tryptic peptides
(1 pL) derived from each protein. MALDI-MS(/MS) spectra were recorded
using an UltrafleXtreme TOF mass spectrometer with a reflector voltage
and a detector voltage of 21 and 17 kV, respectively, as reported.62 (link),63 (link) The Mascot search algorithm v2.0.04 (updated on December 9, 2019;
Matrix Science, London, UK) was used for searching peptide masses.
The identified proteins were assessed for a Mascot score > 56 and p < 0.05.
3
FOXO3-DBD Binding Assay with FoxP3 Probes
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Fluorescence-labeled, double-stranded 100 nM FoxP3 or FoxP3-mut oligonucleotides (FoxP3 forward: 5′-AGCAAAGTTGTTTTTGATAATG-3′, reverse: 5′-CATTATCAAAAACAACTTTGCT-3′; FoxP3-mut forward: 5′-AGCAAAGTTGGGGTTGATAATG-3′, reverse: 3′-CATTATCAACCCCAACTTTGCT-5′; Microsynth, Balgach, Switzerland) [35 (link), 36 (link)] were incubated with 1 µM recombinant FOXO3-DBD protein and CBX in assay buffer (20 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, 5% glycerol) for 30 min at room temperature. For the cell-based FAM-EMSA, 30 µg whole-cell extracts of SH-EP/FOXO3 cells treated with 50 nM 4OHT in combination with CBX were incubated with fluorescence-labeled, double-stranded FoxP3 oligonucleotides (100 nM) in assay buffer for 30 min at room temperature.
The samples were resolved on a 5% polyacrylamide gel in 0.5× TBE running buffer (45 mM Tris-HCL (pH 8.3), 45 mM boric acid, 1.3 mM EDTA). The fluorescence signal was analyzed with the Typhoon 9410 scanner (GE Healthcare, Vienna, Austria).
The samples were resolved on a 5% polyacrylamide gel in 0.5× TBE running buffer (45 mM Tris-HCL (pH 8.3), 45 mM boric acid, 1.3 mM EDTA). The fluorescence signal was analyzed with the Typhoon 9410 scanner (GE Healthcare, Vienna, Austria).
4
DNA Polymerase Activity Assay
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DNA-dependent polymerization was assayed on a template/primer substrate (obtained by hybridization of oligonucleotides Cy5P and T33, see Table 1 ), on 5-nt gapped molecules (obtained by hybridization of oligonucleotides Cy5P, T33 and either DOH or DowP, which contains a 5′-phosphate) and on 1-nt gapped molecules (obtained by hybridization of oligonucleotides Cy5P, T29X and DowP). The incubation mixture (12.5 μl) contained 50 mM Tris-HCl pH 7.5, 1.25 mM MgCl2, 1 mM DTT, 4% (v/v) glycerol, 0.1 mg/ml BSA, 25 nM of the hybrid shown in each case, and the indicated concentrations of PgVPolX (or 2 µl of each of the PgVPolX containing fractions from the glycerol gradient) and nucleotides. After incubation for 5 min at 30 °C the reactions were stopped by adding EDTA to 10 mM. Samples were analyzed by 7 M urea-20% PAGE and visualized using a Typhoon 9410 scanner (GE Healthcare).
5
Quantitative RNA Analysis by Northern Blotting
6
Western Blot Protein Analysis Protocol
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Forty-height hours after transfection, cells were rinsed twice with PBS and then harvested in PBS containing protease inhibitors (cOmplete tablet, Roche). The cells were lysed in PBS containing 1% Igepal and protease inhibitors for 30 min on ice. The detergent lysates were then clarified by centrifugation (14,000 ×g, 30 min, 4 °C). Proteins were separated by SDS-PAGE on 3–8% Tris-Acetate or 4–12% Bis-Tris gels and then transferred to polyvinylidene fluoride membranes. After blocking in TBS buffer (10 mM Tris, pH 7.4, 500 mM NaCl. 0.5% Igepal, 10% goat serum and 3% BSA), the membranes were incubated with primary antibody overnight. The protein-Ab complexes were then labelled with a horseradish peroxidase-conjugated secondary Ab (Sigma-Aldrich) for 1 h at room temperature and detected using the enhanced ECL Plus reagent (GE Healthcare) visualized with a Typhoon 9410 scanner (GE Healthcare). Quantification of immunoblot bands was performed with ImageQuant software (GE Healthcare) or Image J.
7
Protein Expression Quantification
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Cells were harvested, and total soluble proteins were run on polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blotted for relevant proteins using specific primary antibodies (as described for each experiment). Secondary antibodies were FITC or horseradish peroxidase conjugated, and fluorescence was detected using a Typhoon 9410 scanner (GE Healthcare Life Sciences, Piscataway, NJ) or with enhanced chemiluminescence detection reagent. Quantification of the digital images obtained was performed using ImageQuant 5.2 software (GE Healthcare Life Sciences).
8
Western Blot Analysis of Calcium Channel Subunits
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At 48 h after transfection, cells were rinsed twice with
phosphate-buffered saline (PBS) and then harvested in PBS containing protease
inhibitors (Complete tablet from Roche). The cells were lysed in PBS, 1% Igepal
and protease inhibitors for 30 min on ice. The detergent lysates were
then clarified by centrifugation (14,000 g, 30 min,
4 °C). Proteins were separated by SDS–PAGE on
3–8% Tris-acetate
or 4–12% Bis-Tris gels and then transferred to polyvinylidene
fluoride membranes. After blocking in Tris-buffered saline buffer
(10 mM Tris, pH
7.4, 500 mM NaCl.
0.5% Igepal, 10% goat serum and 3% BSA), the membranes were incubated with
primary antibody overnight. The protein–Ab complexes were then
labelled with a horseradish peroxidase-conjugated secondary Ab (1:3,000
Sigma-Aldrich) for 1 h at room temperature and detected using the
enhanced ECL Plus reagent (GE Healthcare) visualized with a Typhoon 9410 scanner
(GE Healthcare). Quantification of immunoblot bands was performed with
ImageQuant software (GE Healthcare). The following Abs were used: rabbit
anti-CaV2.2
(1:500)52 (link), rabbit anti-CaVβ1b (1:500) and
mouse anti-CaVα2δ-1
(1:3,000, D219, Sigma).
phosphate-buffered saline (PBS) and then harvested in PBS containing protease
inhibitors (Complete tablet from Roche). The cells were lysed in PBS, 1% Igepal
and protease inhibitors for 30 min on ice. The detergent lysates were
then clarified by centrifugation (14,000 g, 30 min,
4 °C). Proteins were separated by SDS–PAGE on
3–8% Tris-acetate
or 4–12% Bis-Tris gels and then transferred to polyvinylidene
fluoride membranes. After blocking in Tris-buffered saline buffer
(10 mM Tris, pH
7.4, 500 mM NaCl.
0.5% Igepal, 10% goat serum and 3% BSA), the membranes were incubated with
primary antibody overnight. The protein–Ab complexes were then
labelled with a horseradish peroxidase-conjugated secondary Ab (1:3,000
Sigma-Aldrich) for 1 h at room temperature and detected using the
enhanced ECL Plus reagent (GE Healthcare) visualized with a Typhoon 9410 scanner
(GE Healthcare). Quantification of immunoblot bands was performed with
ImageQuant software (GE Healthcare). The following Abs were used: rabbit
anti-CaV2.2
(1:500)52 (link), rabbit anti-CaVβ1b (1:500) and
mouse anti-CaVα2δ-1
(1:3,000, D219, Sigma).
9
In vitro tRNA 3'-CCA Addition Assay
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Internally labeled tRNAs were in vitro transcribed from cloned tRNA sequences as described [92 (link),93 ]. Varying amounts of CCA1 or CCA2 enzyme were incubated with 5 pmol tRNA and a mix of 1 mM NTPs (equimolar amounts of ATP, CTP, GTP and UTP) for indicated times at 20 °C in 30 mM HEPES/KOH (pH 7.6), 30 mM KCl, 2 mM DTT and 6 mM MgCl2. Reaction products were ethanol precipitated, separated by denaturing polyacrylamide gel electrophoresis and visualized on a Typhoon 9410 Scanner (GE Healthcare, Chicago, IL, USA). For high-throughput sequencing analysis, up to ~500 pmol of phosphodiesterase-treated tRNA of D. discoideum was used with adjusted limited amounts of enzyme to avoid saturating conditions.
10
In vitro activity assay of tRNA nucleotidyltransferases
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For in vitro activity tests of the A. castellanii tRNA nucleotidyltransferases, in vitro transcribed S. cerevisiae tRNAPhe or tRNAPhe-CC (α-32P-ATP-labeld) was produced as described (Mörl and Hartmann 2014 ; Schürer et al. 2002 ). To ensure proper three-dimensional folding of the tRNAs, transcripts were heated up to 65 °C for 5 min and cooled down prior to reaction. Reaction conditions were as following: 5 pmol tRNA and varying amounts of enzyme (1–50 ng) were incubated with 1 mM NTP-mix (equimolar amounts) or 0.1 mM ATP or 0.1 mM CTP in 30 mM HEPES/KOH (pH 7.6), 30 mM KCl, 2 mM DTT, and 6 mM MgCl2 for 30 min at 30 °C. Reaction was stopped by ethanol precipitation, products were dissolved in loading dye, and analyzed by denaturing PAGE and autoradiography using a Typhoon 9410 scanner (GE Healthcare). For each enzyme, at least three independent experiments were performed.
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