Typhoon variable mode imager

Manufactured by Cytiva

Sourced in United States

The Typhoon Variable Mode Imager is a versatile imaging system designed for a wide range of applications in life science research. It offers high-resolution, quantitative imaging capabilities for various fluorescent and chemiluminescent detection methods. The system can be used to analyze a variety of sample types, including gels, blots, and microarrays.

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Lab products found in correlation

Penicillin, by Thermo Fisher Scientific (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions) Facscalibur, by BD (1 mentions) Novex chemiluminescent substrate reagent kit, by Thermo Fisher Scientific (1 mentions) Novex tris glycine gradient gels, by Thermo Fisher Scientific (1 mentions) 0.2 μm pvdf membrane, by Thermo Fisher Scientific (1 mentions) Imiglucerase, by Sanofi (1 mentions) Mini protean tgx gel, by Bio-Rad (1 mentions) Bradford assay, by Bio-Rad (1 mentions) Gelred, by Biotium (1 mentions)

13 protocols using typhoon variable mode imager

Quantifying Glucocerebrosidase Activity in Cells

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Cell homogenates were incubated with MDW941 (100 nM) in McIlvaine buffer [150 mM citrate-Na₂HPO₄ (pH 5.2), 0.2% (w/v) sodium taurocholate, 0.1% (v/v) Triton X-100] for 30 min at 37°C and subjected to SDS-PAGE. Fluorescent GBA on slab gels was visualized using a fluorescence scanner (Typhoon variable mode imager, Amersham Biosciences) (8 (link)).
Intact fibroblasts were incubated with MDW941 (5 nM) for 72 h, and subsequently the culture medium was collected and the cells were harvested. Detection of fluorescent GBA in the culture medium was performed upon capture of the enzyme from 1 ml medium using monoclonal antibody 8E4 immobilized to Sepharose beads (25 (link)).
For fluorescence-activated cell sorting (FACS), fibroblasts and macrophages were incubated with MDW933 (50 nM) for 5 h in the medium (8 (link)). In the case of white blood cells, leukocytes were collected from freshly drawn blood washed with 0.8% (w/v) ammonium chloride solution and lysing the remaining erythrocytes. Leukocytes were next incubated with MDW933 (100 nM) for 30 min in phosphate buffered saline containing 1% (w/v) BSA. FACS analysis was performed with a FACSCalibur (B.D. Bioscience), λ_ex 488 nm, λ_em 530 nm (bandpass filter 30 nm) (8 (link)).

Gaspar P., Kallemeijn W.W., Strijland A., Scheij S., Van Eijk M., Aten J., Overkleeft H.S., Balreira A., Zunke F., Schwake M., Sá Miranda C, & Aerts J.M. (2014). Action myoclonus-renal failure syndrome: diagnostic applications of activity-based probes and lipid analysis. Journal of Lipid Research, 55(1), 138-145.

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Activity-Based Protein Profiling of Retaining Glycosidases

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All pre-incubations and ABP labeling-reactions occurred for 30 min at 37 °C. Total lysates (50 μg), medium (500 μg) or purified protein preparations (5 μg) were labeled with 1 μM JJB256 1 or JJB244 2, dissolved in 150 mM McIlvaine buffer, pH 4.5, incubating for 30 min at 37 °C. For ABPP, protein preparations were pre-incubated with compounds 4 (100 μM), 6 (100 μM), 7–13 (1 or 5 mM, specified in the main text) prior to the addition of 100 nM fluorescent ABPs. Influence of pH on ABP labeling involved pre-incubation at pH 3–10 prior to addition of 100 nM ABP 1. Direct labeling of retaining β-glucosidases occurred at pH 5.0 in conjunction with 100 nM ABP JJB75. Samples were then denatured with 5× Laemmli buffer (50% (v/v) 1 M Tris–HCl, pH 6.8, 50% (v/v) 100% glycerol, 10% (w/v) DTT, 10% (w/v) SDS, 0.01% (w/v) bromophenol blue), boiled for 4 min at 100 °C, and separated by gel electrophoresis on 10% (w/v) SDS-PAGE gels running continuously at 90 V.^{13 (link),14 (link)} Wet slab-gels were scanned on fluorescence using a Typhoon Variable Mode Imager (Amersham Biosciences) using λ_EX 488 nm and λ_EM 520 nm (band pass filter 40 nm) for green fluorescent JJB256 1 and λ_EX 532 nm and λ_EM 610 nM (band pass filter 30 nm) for red fluorescent JJB244 2 and JJB75.

Jiang J., Kallemeijn W.W., Wright D.W., van den Nieuwendijk A.M., Rohde V.C., Folch E.C., van den Elst H., Florea B.I., Scheij S., Donker-Koopman W.E., Verhoek M., Li N., Schürmann M., Mink D., Boot R.G., Codée J.D., van der Marel G.A., Davies G.J., Aerts J.M, & Overkleeft H.S. (2015). In vitro and in vivo comparative and competitive activity-based protein profiling of GH29 α-l-fucosidases †Electronic supplementary information (ESI) available: Experimental part including synthesis procedures, characterization data, copies of NMR spectra of new compounds, supporting biological figures and crystallographic information. See DOI: 10.1039/c4sc03739a. Chemical Science, 6(5), 2782-2789.

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Western Blot Analysis of Protein Lysates

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Protein lysates from cell and tissues were lysed and homogenized (tissues) in Mammalian Protein Extraction Reagent (M-PER) lysis buffer (Cat. no. 78501; Thermo Fisher Scientific) supplemented with cOmplete protease inhibitor tablets (Cat. no. 1183617000; MilliporeSigma). Approximately 50 μg of lysate were electrophoretically resolved on 4–20% Novex Tris-glycine gradient gels (Cat. no. XV04200PK20; Thermo Fisher Scientific). Proteins were then transferred to 0.2-μm PVDF membranes (Cat. no. 88520; Thermo Fisher Scientific) and incubated overnight at 4°C with gentle rocking in primary antisera diluted 1:1,000 in 5% bovine serum albumin (Cat. no. A30075; RPI Corp)/1xTBS-Tween (Cat. no. IBB-581X; Boston BioProducts). Membranes were washed three times in 1xTBS-Tween for 5 min each and then incubated in secondary antisera (1:2,000 dilution) for 1 h at room temperature. Membranes were then washed four times in 1× TBS-Tween for 15 min each before the addition of the Novex Chemiluminescent Substrate Reagent Kit (Cat. no. WP20005; Thermo Fisher Scientific). Membranes were exposed onto PerfectFilm audioradiography film (Cat. no. B581; GenHunter) and developed on a Typhoon Variable Mode Imager (Amersham Pharmacia). Western blot densitometry was performed using open-source ImageJ software.

Samani A., Hightower R.M., Reid A.L., English K.G., Lopez M.A., Doyle J.S., Conklin M.J., Schneider D.A., Bamman M.M., Widrick J.J., Crossman D.K., Xie M., Jee D., Lai E.C, & Alexander M.S. (2022). miR-486 is essential for muscle function and suppresses a dystrophic transcriptome. Life Science Alliance, 5(9), e202101215.

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Fluorescent Assay for GCase Activity

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Florescent activity-based probes specific for GCase (MDW933) were synthesized at the Imaging Probe Development Center (National Heart Lung and Blood Institute, Bethesda, MD, USA) as previously described (Witte et al., 2010 (link)). Total protein concentration of each sample was measured using a Bradford assay according to the manufacturer's guidelines (Bio-Rad Laboratories). Cell homogenate was incubated with 1 μM of green fluorescent MDW933 probe in citrate phosphate buffer (pH 5.4) at 37°C for 90 min. Samples were analyzed on a 4-20% Mini-PROTEAN^® TGX™ gel (Bio-Rad Laboratories) using 1.2 μM imiglucerase (Genzyme, Cambridge, MA, USA) with 1 μM MDW933 probe as a control. A Typhoon Variable Mode Imager (Amersham Biosciences, Piscataway, NJ, USA), set to excitation wavelength (λ_ex)=488 nm and emission wavelength (λ_em)=520 nm, was used to measure the fluorescent signal in the gel.

Westbroek W., Nguyen M., Siebert M., Lindstrom T., Burnett R.A., Aflaki E., Jung O., Tamargo R., Rodriguez-Gil J.L., Acosta W., Hendrix A., Behre B., Tayebi N., Fujiwara H., Sidhu R., Renvoise B., Ginns E.I., Dutra A., Pak E., Cramer C., Ory D.S., Pavan W.J, & Sidransky E. (2016). A new glucocerebrosidase-deficient neuronal cell model provides a tool to probe pathophysiology and therapeutics for Gaucher disease. Disease Models & Mechanisms, 9(7), 769-778.

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Lipid Separation by TLC Imaging

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The extracted lipids were resuspended in chloroform and resolved by TLC (preactivated by heating at 90°C for 1 h) with a running solvent (45:35:8:2 chloroform: methanol: water: 25% ammonia). Plates were air-dried and imaged on a Typhoon Variable Mode Imager (Amersham Biosciences).

Ghosh A., Venugopal A., Shinde D., Sharma S., Krishnan M., Mathre S., Krishnan H., Saha S, & Raghu P. (2023). PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase. Life Science Alliance, 6(9), e202301920.

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Lipid Analysis by TLC Imaging

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Extracted lipids were resuspended in chloroform and resolved by TLC (preactivated by heating at 90°C for 1 h) with a running solvent (45:35:8:2 chloroform: methanol:water:25% ammonia). Plates were air dried and imaged on a Typhoon Variable Mode Imager (Amersham Biosciences).

Ghosh A., Sharma S., Shinde D., Ramya V, & Raghu P. (2019). A novel mass assay to measure phosphatidylinositol-5-phosphate from cells and tissues. Bioscience Reports, 39(10), BSR20192502.

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DNA-Protein Binding Assay

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The DNA substrates (Integrated DNA Technologies) (Supplementary Table S1) were resuspended in binding buffer (50 mM Tris–HCl pH 8, 50 mM NaCl, 10% (v/v) glycerol) and incubated with and without the different purified proteins at the indicated concentrations in a final volume of 10 μl. After gentle mixing, samples were incubated for 30 min at 4°C and then loaded onto a 0.4× TBE, 0.7% agarose gel. Electrophoresis was carried out in 0.4× TBE at 100 V and 4°C for 90 min. Finally, the gel was either stained with GelRed (Biotium), washed in 0.4× TBE and imaged with UV illumination or directly imaged in a Typhoon Variable Mode Imager (Amersham Biosciences) using a 480-nm laser and recording the emission at 520 nm for the 6FAM-labeled substrates. Gels were then stained with Quick Coomassie Stain (Protein Ark) for visualization of protein bands.

Coloma J., Gonzalez-Rodriguez N., Balaguer F.A., Gmurczyk K., Aicart-Ramos C., Nuero Ó.M., Luque-Ortega J.R., Calugaru K., Lue N.F., Moreno-Herrero F, & Llorca O. (2023). Molecular architecture and oligomerization of Candida glabrata Cdc13 underpin its telomeric DNA-binding and unfolding activity. Nucleic Acids Research, 51(2), 668-686.

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Visualizing Active Endogenous GBA1 and Cysteine Cathepsins

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ABP-MDW941/Inhibody Red [34 (link)] was used (1 nM for 16 h; synthesized in reference [34 (link)]) to label active endogenous GBA1 molecules in RAW cells. Images were taken with a confocal SP5 Leica with a 63x objective using an excitation wavelength of 561 nm. For cysteine cathepsin labeling, ABP DCG-04 [35 (link)] was added (500 nM for 2 h; synthesized in reference [35 (link)]) to cells. After rinsing in PBS, cell homogenates were prepared in KPi lysis buffer (25 mM K₂HPO₄/KH₂PO₄, pH 6.5, 0.1% [v:v] Triton X-100) supplemented with protease inhibitors. After protein separation with SDS-PAGE (10%), fluorescence was subsequently monitored in wet slab gels with a Typhoon Variable Mode Imager (Amersham Biosciences) using λex 488 nm and λem 520 nm (bandpass 40).

Tol M.J., van der Lienden M.J., Gabriel T.L., Hagen J.J., Scheij S., Veenendaal T., Klumperman J., Donker-Koopman W.E., Verhoeven A.J., Overkleeft H., Aerts J.M., Argmann C.A, & van Eijk M. (2018). HEPES activates a MiT/TFE-dependent lysosomal-autophagic gene network in cultured cells: A call for caution. Autophagy, 14(3), 437-449.

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Telomere Length Measurement Protocol

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Gels from all 17 species were imaged on a phosphor screen with a Typhoon Variable Mode Imager (Amersham Biosciences, Buckinghamshire, UK) to visualize telomeres. The amount of radioactive signal (optical density, OD) in each lane corresponds with the amount of telomere at that position on the gel (i), and was quantified by densitometry in ImageJ (v. 1.51). Background signal from nonspecific binding of the radioactive probe was subtracted from all OD measures. The specific molecular markers on each gel differed because gel conditions were optimized based upon a species' particular telomere distribution (1 kb DNA ladder (1–10 kb); 1 kb plus DNA ladder (1–12 kb); Lambda DNA/EcoR1 +HindIII (1–21 kb); 1 kb DNA extension ladder (1–4 0 kb), Invitrogen; λ DNA Monocut (2–49 kb) New England Biolabs; DNA Marker XV (2–49 kb), Roche; PFG Marker 1 (15–200 kb), New England Biolabs). However, regardless of the molecular marker used, the distance each band of the molecular marker migrated (i) was plotted against the molecular weight in kilobases and converted into molecular weights (L) using a three-parameter log-linear function. The mean TRF length (called mean telomere length hereafter for simplicity) for each individual was calculated using: mean TRF = ∑(ODi * LI)/∑(ODi), where ODi is the densitometry output at position i, and Li is the length of the DNA (kB) at position I [52 (link)].

Tricola G.M., Simons M.J., Atema E., Boughton R.K., Brown J.L., Dearborn D.C., Divoky G., Eimes J.A., Huntington C.E., Kitaysky A.S., Juola F.A., Lank D.B., Litwa H.P., Mulder E.G., Nisbet I.C., Okanoya K., Safran R.J., Schoech S.J., Schreiber E.A., Thompson P.M., Verhulst S., Wheelwright N.T., Winkler D.W., Young R., Vleck C.M, & Haussmann M.F. (2018). The rate of telomere loss is related to maximum lifespan in birds. Philosophical Transactions of the Royal Society B: Biological Sciences, 373(1741), 20160445.

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Quantitative Fluorescent Protein Assay

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In a 24-well plate, 1 μL of 200x inhibitor concentration was added to approximately 10⁵ A549+ACE2 cells in 200 μL medium containing 1% DMSO and incubated for 1 h at 37 °C. 1 μL of BMV109 was added at a final concentration of 1 μM and incubated for 1h. Medium was removed, and cells were detached from culture plate by incubating with 100 μL 0.05 % Trypsin, 0.5 mM EDTA solution for 10 min at 37 °C. Cells were spun down, washed twice with PBS and lysed by four succeeding freeze-thaw cycles via submersion of Eppendorf tubes in a 37 °C water bath and liquid nitrogen respectively. Protein concentration of lysate was determined using BCA assay, Laemmli’s sample buffer was added at 4-fold dilution and samples were boiled for 5 min before running them on 15% SDS-PAGE gel. In-gel detection of fluorescently labeled proteins was performed directly by scanning the wet gel slabs on the Typhoon Variable Mode Imager (Amersham Biosciences) using Cy5 settings (λ_ex 650 nm, λ_em 670 nm). Densitometric analysis of protein bands on gels was performed using ImageJ (v1.52p).

Steuten K., Kim H., Widen J.C., Babin B.M., Onguka O., Lovell S., Bolgi O., Cerikan B., Neufeldt C.J., Cortese M., Muir R.K., Bennett J.M., Geiss-Friedlander R., Peters C., Bartenschlager R, & Bogyo M. (2021). Challenges for targeting SARS-CoV-2 proteases as a therapeutic strategy for COVID-19. ACS infectious diseases, 7(6), 1457-1468.

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