typhoon trio scanner, by Cytiva - Product details

1

Reverse Transcription Reaction Monitoring

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Reverse transcription reactions containing 50mM Tris-HCl (pH 8.0), 50mM KCl, 6mM MgCl₂, 5mM β-me, 200nM vRNA-tRNA complex, and 3μM RT were pre-incubated at 37°C for 4 minutes. vRNA-tRNA complexes consisted of the vRNA construct for smFRET heat annealed to synthetic tRNA_Lys³ with a 5’ Cyanine3 label (TriLink). Reactions were initiated by addition of a dNTP mixture to a final concentration of 100μM per nucleotide. Reactions for the ladder lanes were identical except for the addition of 25μM ddNTP for one nucleotide species in each corresponding sequencing lane. Reactions were quenched at time points with 1:1 reaction:buffer volume of loading buffer containing 50mmM Tris-HCl pH 8.0, 50mM EDTA, and 90% formamide.
Reactions were then heated for 5 min at 95 °C, loaded onto an 8.5% polyacrylamide gel which had been pre-run at 100W for 2 hours. The gel was run at 120W for 3 hours before imaging with a Typhoon Trio scanner (Amersham Biosciences).

Coey A.T., Larsen K.P., Choi J., Barrero D.J., Puglisi J.D, & Puglisi E.V. (2018). Dynamic Interplay of RNA and Protein in the Human Immunodeficiency Virus-1 Reverse Transcription Initiation Complex. Journal of molecular biology, 430(24), 5137-5150.

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2

Comparative Proteomics via 2D-DIGE

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Two-dimensional DIGE was performed at Applied Biomics (Hayward, CA, USA) following typical methods [19 ,20 (link)]. Briefly, cell lysates, were denatured by equal volume addition of lysis buffer containing 7M urea, 2M thio urea, 4% 3-((3-cholamidopropyl)dimethyl ammonio)-1-propanesulfonate(CHAPS) followed by addition of 30 mM Tris-HCl, pH 8.8, at a 5:1 volume ratio lysis buffer: plasma. Lysate samples were normalized using total protein as determined by Lowry protein estimation method. Next, samples were labeled with CyDye DIGE fluors developed for fluorescence 2D-DIGE technology (Cy3 and Cy5, GE Healthcare, CT, USA) and incubated in dark on ice, 30 min. The labeled samples were then subjected to isoelectric focusing (IEF) on a 13-cm precast non-linear immobilized pH gradient strip (pH 3-10, Amersham Biosciences, Buckinghamshire, UK) using an Amersham Pharmacia IPGPHOR unit with a power supply (EPS3501XL) in gradient mode. Next, the samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) in the second dimension based on size. The gels were scanned using Typhoon Trioscanner (Amersham Biosciences) and fluorescent dye signals corresponding to individual samples converted to black and white images for analysis using Image Quant and DeCyder software (Amersham Biosciences).

Banerjee H., Hyman G., Evans S., Manglik V., Gwebu E., Banerjee A., Vaughan D., Medley J., Krauss C., Wilkins J., Smith V., Banerji A, & Rousch J. (2014). Identification of the Transmembrane Glucose Regulated Protein 78 as a Biomarker for the Brain Cancer Glioblastoma Multiforme by Gene Expression and Proteomic Studies. Journal of membrane science & technology, 4(1), 1000126.

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3

Protoplast Formation and Protein Analysis

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B. subtilis was grown as described above. For each sample 400 µl cells were pelleted (5,000xg, 1 min, 37°C) and resuspended in 20 µl MSMNB (500 mM sucrose, 20 mM MgCl₂, 20 mM malic acid, 13 g l^-1 nutrient broth, pH 7.5) and 15 nm SNAP-CellCellCell TMR-Star (NEB) final concentration was added. Lysozyme was added to a final concentration of 2 mg ml^-1 from a 10 mg ml^-1 stock solved in MSMNB, cells were incubated at 37°C without shaking and protoplastation was followed microscopically. ProteinaseK was added to a final concentration of 0.1 mg ml^-1 from a 1 mg ml^-1 stock solved in MSMNB. ProteinaseK digestion was stopped by addition of 10 mM phenylmethanesulfonylfluoride (PMSF) final concentration. Samples were mixed with 10 µl 4x SDS-PAGE loading dye, cooked for 20 minutes at 95°C and analysed via SDS-PAGE. In gel-fluorescence was analysed using a Typhoon Trio scanner (Amersham Biosciences) using a 532 nm laser and a 580 nm emission filter. Quantification of fluorescence was performed using ImageJ [33 (link)].

Bach J.N, & Bramkamp M. (2015). Dissecting the Molecular Properties of Prokaryotic Flotillins. PLoS ONE, 10(1), e0116750.

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4

Comparative Proteomics via 2D-DIGE

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Two-dimensional DIGE was performed at Applied Biomics (Hayward, CA, USA) following typical methods [19 ,20 (link)]. Briefly, cell lysates, were denatured by equal volume addition of lysis buffer containing 7M urea, 2M thio urea, 4% 3-((3-cholamidopropyl)dimethyl ammonio)-1-propanesulfonate(CHAPS) followed by addition of 30 mM Tris-HCl, pH 8.8, at a 5:1 volume ratio lysis buffer: plasma. Lysate samples were normalized using total protein as determined by Lowry protein estimation method. Next, samples were labeled with CyDye DIGE fluors developed for fluorescence 2D-DIGE technology (Cy3 and Cy5, GE Healthcare, CT, USA) and incubated in dark on ice, 30 min. The labeled samples were then subjected to isoelectric focusing (IEF) on a 13-cm precast non-linear immobilized pH gradient strip (pH 3-10, Amersham Biosciences, Buckinghamshire, UK) using an Amersham Pharmacia IPGPHOR unit with a power supply (EPS3501XL) in gradient mode. Next, the samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) in the second dimension based on size. The gels were scanned using Typhoon Trioscanner (Amersham Biosciences) and fluorescent dye signals corresponding to individual samples converted to black and white images for analysis using Image Quant and DeCyder software (Amersham Biosciences).

Banerjee H., Hyman G., Evans S., Manglik V., Gwebu E., Banerjee A., Vaughan D., Medley J., Krauss C., Wilkins J., Smith V., Banerji A, & Rousch J. (2014). Identification of the Transmembrane Glucose Regulated Protein 78 as a Biomarker for the Brain Cancer Glioblastoma Multiforme by Gene Expression and Proteomic Studies. Journal of membrane science & technology, 4(1), 1000126.

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5

Protein kinase activity assay

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Proteins were incubated with 100 nCi [γ³²-P]ATP (PerkinElmer) in 50 mM Tris⋅HCl, pH 8, buffer containing 10 mM MgCl₂, 5 mM MnCl₂, and 20 μM cold ATP at room temperature for 1 h. The samples were then separated on SDS/PAGE gels, which were exposed overnight on phosphor plates (Molecular Dynamics). The phosphor plates were scanned with the typhoon TRIO scanner (Amersham Biosciences).

Wong J.E., Nadzieja M., Madsen L.H., Bücherl C.A., Dam S., Sandal N.N., Couto D., Derbyshire P., Uldum-Berentsen M., Schroeder S., Schwämmle V., Nogueira F.C., Asmussen M.H., Thirup S., Radutoiu S., Blaise M., Andersen K.R., Menke F.L., Zipfel C, & Stougaard J. (2019). A Lotus japonicus cytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation. Proceedings of the National Academy of Sciences of the United States of America, 116(28), 14339-14348.

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6

Quantitative Proteomic Analysis of Differential Protein Abundance

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Gels were run in triplicate, scanned, and analyzed as described previously [44 (link)]. To summarize, scanning was performed using a Typhoon Trioscanner (Amersham BioSciences) following the manufacturer’s protocol. Scanned images were processed using Image Quant software (Amersham BioSciences, v.5.0). Protein abundance was assessed by differential in-gel analysis (DIA). The quantitative analysis of protein spots was performed using DeCyder software (Amersham Biosciences, v.6.5). Quantitative comparisons were calculated between samples run at the same time and pair-wise volume ratios were calculated for each protein spot and used to determine relative fold changes ratios. Fold changes were log₂ transformed. Student’s t-test was performed using the log2 normalized average spot volume ratios for all spots detected from the three replicate experiments. Only statistically significant results (P<0.05), and differentially abundant proteins with a ratio > 2-fold (log2 of 1) difference in one condition (increase or decrease in abundance), were chosen for mass spectrometry.

Die J.V., Arora R, & Rowland L.J. (2017). Proteome dynamics of cold-acclimating Rhododendron species contrasting in their freezing tolerance and thermonasty behavior. PLoS ONE, 12(5), e0177389.

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7

Membrane Protein Isolation and Analysis

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Cells were cultivated as described above. In the early and late exponential growth phase (OD₆₀₀ ~0.08 and ~0.7, respectively), cells were harvested by centrifugation (7,000 × g, 15 min, 4°C). The pellet was resuspended in TG buffer (50 mM Tris/HCl pH 8.0, 10% (v/v) glycerol) supplemented with lysozyme, DNase and PMSF (0.5 mM) and thereby concentrated 200 fold. After incubation at 37°C for 15 min, membranes were collected by ultracentrifugation (100,000 × g, 15 min, 4°C). The pellet was resuspended in TG buffer and diluted when indicated. Samples were mixed with buffer [200 mM Tris/HCl (pH 8.8), 20% (v/v) glycerol, 5 mM EDTA (pH 8.0), 0.02% (w/v) bromphenol blue (aliquots of 700 μl)], and before use 200 μl 20% (w/v) SDS and 100 μl 0.5 M DTT (Drew et al., 2006 (link)) was added. SDS-PAGE was performed according to Laemmli (Laemmli, 1970 (link)) and run at 150 V for 2 h in the dark to avoid bleaching of the fluorophore. In gel-fluorescence was analyzed using the Typhoon Trio scanner (Amersham Biosciences) with 488 nm laser and 526 nm emission filter. Quantification of fluorescence was performed using ImageQuant (GE Healthcare, München, Germany).

Lorenz N., Shin J.Y, & Jung K. (2017). Activity, Abundance, and Localization of Quorum Sensing Receptors in Vibrio harveyi. Frontiers in Microbiology, 8, 634.

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8

Cohesin ATPase Activity Assay

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Cohesin complexes were incubated in 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 5% glycerol, 0.1 mg/ml BSA, 10 nM [γ‐³²P]‐ATP, and 50 μM non‐radiolabeled ATP. Reactions were incubated at 32°C and stopped by adding 1% SDS and 10 mM EDTA. Reaction products were separated on polyethyleneimine plates (EMD Biosciences) by thin‐layer chromatography using 0.75 M KH₂PO₄ (pH 3.4) and analyzed by phosphor imaging with a Typhoon Trio Scanner (Amersham).

Davidson I.F., Goetz D., Zaczek M.P., Molodtsov M.I., Huis in 't Veld P.J., Weissmann F., Litos G., Cisneros D.A., Ocampo‐Hafalla M., Ladurner R., Uhlmann F., Vaziri A, & Peters J. (2016). Rapid movement and transcriptional re‐localization of human cohesin on DNA. The EMBO Journal, 35(24), 2671-2685.

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9

Monitoring α-Synuclein Aggregation Inhibition

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Solutions of 70 μM monomer and 5% seeds of α-syn alone or in the presence of 0.5 μM Hsp70, or 0.5 μM DNAJB6, or 0.15 μM DNAJB6, or 0.35 μM Hsp70 and 0.15 μM DNAJB6 in 50 mM Tris pH 7.4, 150 mM KCl, 5 mM MgCl₂, 0.01% NaN₃, 5 mM ATP were incubated for 15 h at 37 °C in quiescence. Samples were then centrifuged at 120,000 rpm at 10 °C for 1 h using a TLA-120.2 Beckman rotor. Samples of 3 μl of the supernatants were then spotted on a 0.45 μm pore size nitrocellulose membrane (Amersham Bioscience, Little Chalfont, UK) and let dry for 1 h RT.
The membrane was blocked in PBS + 5% (w/v) BSA for 1 h RT and then incubated in PBS + 5% (w/v) BSA + 1:2000 mouse anti α-syn polyclonal antibody (Transduction Laboratories, Lexington, KY, USA) ON at 4 °C. After incubation with primary antibody, it was washed in PBS + 0.02% Tween-20 three times, 15 min each time, and incubated in PBS + 5% (w/v) BSA + 0.02% Tween-20 + 1:2000 goat anti-mouse Alexa Fluor-488 secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at RT. Finally, the membrane was washed 3 times in PBS + 0.02% Tween-20 and the fluorescence reviled by utilizing a Typhoon Trio scanner (Amersham Bioscience, Little Chalfont, UK).

Aprile F.A., Källstig E., Limorenko G., Vendruscolo M., Ron D, & Hansen C. (2017). The molecular chaperones DNAJB6 and Hsp70 cooperate to suppress α-synuclein aggregation. Scientific Reports, 7, 9039.

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10

Immunoblot analysis of amyloid oligomers

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In total, 1 μg of monomer or oligomer was deposited onto a 0.2 μm PVDF membrane (Millipore (UK) Ltd., West Lothian, UK) and left to dry at room temperature (RT). The membranes were then blocked (5% (w/v) BSA in PBS, 1 h, RT) and subsequently incubated with A11 or 211 antibody in 5% BSA in PBS (overnight, 4 °C) at 1:5000 and 1:2000 dilutions, respectively. Membranes were washed in PBS + 0.01% Tween-20 (PBST) (3 × 10 min, RT) then incubated with secondary antibody (Alexa Fluor-488 goat anti-mouse for A11, and Alexa Fluor-488 goat anti-rabbit for 211, both 1:5000 (Thermo Fisher Scientific, Waltham, MA, USA)) in PBST (1 h, RT). Following washing in PBST (3 × 10 min, RT), membranes were imaged on a Typhoon Trio scanner and the images analysed using ImageQuant TL v2005 (both Amersham Bioscience, Little Chalfont, UK).

Xu C.K., Castellana-Cruz M., Chen S.W., Du Z., Meisl G., Levin A., Mannini B., Itzhaki L.S., Knowles T.P., Dobson C.M., Cremades N, & Kumita J.R. (2022). The Pathological G51D Mutation in Alpha-Synuclein Oligomers Confers Distinct Structural Attributes and Cellular Toxicity. Molecules, 27(4), 1293.

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Typhoon trio scanner

Lab products found in correlation

12 protocols using typhoon trio scanner

Reverse Transcription Reaction Monitoring

Comparative Proteomics via 2D-DIGE

Protoplast Formation and Protein Analysis

Comparative Proteomics via 2D-DIGE

Protein kinase activity assay

Quantitative Proteomic Analysis of Differential Protein Abundance

Membrane Protein Isolation and Analysis

Cohesin ATPase Activity Assay

Monitoring α-Synuclein Aggregation Inhibition

Immunoblot analysis of amyloid oligomers

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