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ProbesOnline Special Edition: The Most Popular Articles of 2010
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Highly specific, high-quality monoclonal antibodies, unmatched for producing consistent results. Molecular Probes Technology Network Connect with individuals across the world interested in fluorescence imaging and related workflows. Register to win! Register by December 25 and be entered to win a softcover copy of the new Molecular Probes® Handbook, 11th edition. Five winners will be announced and notified on December 30. |
TOP 10 OF 2010
The Qubit® 2.0 Fluorometer — Bring the Power of Fluorescence Quantitation to Your Bench
| what it is The Qubit® 2.0 Fluorometer is the next generation in DNA, RNA, and protein quantitation. Central to the Qubit® Fluorometric Quantitation Platform, this instrument was designed with one thing in mind: to enable you to easily and accurately quantitate your nucleic acid or protein sample, and to help avoid wasted samples or time repeating experiments. what it offers
| how it works The Qubit® 2.0 Fluorometer is an easy-to-use analytical instrument featuring advanced optics, data analysis algorithms, and an intuitive touch screen user interface. The instrument is designed to work seamlessly with Qubit™ assays for DNA, RNA, and protein quantitation. The integrated design of the instrument and the assays makes the Qubit® 2.0 Fluorometer far more sensitive and accurate than UV absorbance; this helps to avoid the repetition of experiments necessitated by inaccurate nucleic acid or protein measurements.
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Applied Biosystems® Attune™ Acoustic Focusing Cytometer — The Latest Advance in Cell-by-Cell Analysis
| what it is By combining the latest advances in acoustic focusing technology with single-cell analysis, the Applied Biosystems® Attune™ Acoustic Focusing Cytometer gives you optimized performance and throughput without sacrificing sensitivity or accuracy. what it offers
| how it works The Attune™ Acoustic Focusing Cytometer uses ultrasound waves to align (focus) cells in the center of the sample capillary before they pass through the optical chamber, resulting in less variable data compared to traditional flow cytometers. The results give you more accurate discrimination of cell populations of interest. The unique design of the Attune™ cytometer also offers remarkable sample rates of up to 1,000 µL/min, over 10 times that of other cytometers, allowing you to collect data faster and to run dilute samples without the need to concentrate them.
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Premo™ Autophagy Sensors — Fluorescence-Based Tools for Visualizing Autophagy
| what they are LC3B plays a critical role in autophagy, and its localization can be used as a general marker for this vital process. Premo™ Autophagy Sensors enable the temporal and spatial visualization of LC3B. what they offer
| how they work Premo™ Autophagy Sensors combine the selectivity of an LC3B–fluorescent protein (LC3B-FP) chimera with the transduction efficiency of BacMam technology, facilitating unambiguous visualization of this protein with an easy one-step protocol. Simply add the BacMam LC3B-FP reagent to cells, incubate overnight for protein expression, then image and analyze. The kits include an LC3B-FP mutant, which serves to indicate whether punctate staining may be due to FP aggregation, and chloroquine, which causes accumulation of autophagosomes.
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Click-iT® DIBO Reagents — Copper-Free Protein Detection in Live Cells
| what they are The use of copper in labeling and detection reactions can be harmful to cells, reduce the fluorescence of certain fluorophores, and impair the activity of some enzymes. Our new Click-iT® DIBO reagents now offer a means of achieving click reactions without copper, enabling live-cell surface labeling of proteins and sugars using click chemistry and gentle conjugation of azide-labeled material. what they offer
| how they work Click chemistry employs a highly specific bioorthogonal reactive chemistry for the in situ labeling of biomolecules. The classic click reaction involves copper-catalyzed triazole formation from an azide and an alkyne, but our new DIBO reagents permit labeling of azide-modified macromolecules without metal catalysts, which enables live-cell detection and prevents protein damage. Nine new products are available for copper-free detection, including DIBO derivatives of Alexa Fluor® dyes, TAMRA dye, and biotin labels, and reactive probes capable of modifying amine, cysteine, and carboxy groups.
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CellLight® Reagents — Specific Targeting of Key Cellular Structures
| what they are CellLight® reagents are fluorescent protein–signal peptide fusions that permit accurate and specific targeting to cellular structures for live-cell imaging applications, or for fixed-cell analyses following formaldehyde-based fixation. Targeted cellular structures include the cytoskeleton, mitochondria, lysosomes, and the secretory and autophagy pathways. what they offer
| how they work CellLight® reagents are powered by BacMam technology, which employs a modified insect cell baculovirus coupled with a mammalian promoter as a vehicle to efficiently deliver and express genes in mammalian cells. The inability of baculoviruses to replicate in mammalian cells renders them safe as research reagents and provides a transient, footprint-free method to label cells. Unlike expression vectors, BacMam reagents enable titratable and reproducible expression and offer high cotransduction efficiency, enabling multiple BacMam reagents to be used in the same cell.
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Virtual Cell Staining Tool — Create Your Own Virtual Cell
| what it is Explore the fascinating world of cellular imaging with this new online tool. Select different combinations of cellular structures and fluorophores to create your version of the perfectly labeled fluorescent cell! what it offers
| how it works Stain your own cell virtually, using our brand-new cell staining simulation tool to develop reproducible results with many of our signature fluorescent dyes. |
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CellTrace™ Violet Cell Proliferation Kit — Characterize 10 Generations of Proliferating Cells
| what it is Amine-reactive cell tracing dyes, such as the new CellTrace™ Violet dye, are known to provide excellent long-term retention in cells. CellTrace™ Violet is a cell-permeant dye that enters live cells, where it is converted to a fluorescent derivative by nonspecific esterases. The resulting succinimidyl ester covalently binds to amine groups in proteins, resulting in long-term dye retention. what it offers
| how it works When a cell labeled with CellTrace™ Violet divides, each daughter cell receives approximately half of the fluorescent label, the next generation receives a quarter, and so on. Analysis of the fluorescence intensities of cells labeled and grown in vivo or in vitro enables determination of the number of generations through which a cell has progressed since the label was applied. The kit allows you to identify dividing cells in biological systems. |
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NovaBright™ SEAP 2.0 Detection Kit — Easier Detection of Secreted Alkaline Phosphatase
| what it is The NovaBright™ Secreted Placental Alkaline Phosphatase (SEAP) 2.0 kit makes detection of SEAP reporter gene activity easier than ever. The procedure has been simplified down to two ready-to-use assays—reagent preparation and sample dilution steps have been eliminated! what it offers
| how it works The kit combines our high-performance alkaline phosphatase substrate, CSPD®, and our next-generation Emerald™ enhancer to provide better assay performance over 5 orders of magnitude of enzyme concentration. Our proprietary buffer formulation minimizes the background due to endogenous phosphatases, enabling the NovaBright™ SEAP 2.0 kit to provide the most sensitive detection with a higher signal-to-noise ratio of SEAP reporter gene activity than other assays of its kind.
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Exposing mtDNA Biogenesis — Combining Click-iT® EdU With Signal Amplification
Detecting Mitochondrial Biogenesis
Mitochondrial biogenesis enables neurons to meet changing energy loads and to redistribute mitochondria throughout the neuron. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication. Traditional methods for monitoring nascent nuclear DNA synthesis follow the incorporation of a nucleoside analog of thymidine into DNA—involving either radioactive 3H-thymidine or BrdU. Direct visualization of mtDNA synthesis can be challenging, due to the weak signal obtained from the small mitochondrial genome (~17 kb); inhibiting nuclear DNA replication does not increase sensitivity.
New Technique
The easy-to-use Click-iT® EdU assay, when combined with Tyramide Signal Amplification (TSA®) technology, enables sensitive and reliable measurement of nascent mtDNA synthesis. The Click-iT® EdU assay is much easier to perform, eliminating the use of radioactivity and avoiding the harsh DNA denaturation step required with the BrdU method, while TSA® technology has been reported to increase detection sensitivity up to 100-fold compared to conventional avidin–biotinylated enzyme complex (ABC) procedures.
TSA® is an enzyme-mediated detection method that uses the catalytic activity of horseradish peroxidase (HRP) to achieve high-density labeling of a target antigen. For the mtDNA detection assay, the target antigen is the fluorescent azide used in the Click-iT® EdU assay to detect DNA containing incorporated EdU. An HRP-conjugated anti-dye antibody is then combined with a tyramide analog that has the same fluorescence emission as the original fluorescent azide.
Mitochondrial biogenesis enables neurons to meet changing energy loads and to redistribute mitochondria throughout the neuron. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication. Traditional methods for monitoring nascent nuclear DNA synthesis follow the incorporation of a nucleoside analog of thymidine into DNA—involving either radioactive 3H-thymidine or BrdU. Direct visualization of mtDNA synthesis can be challenging, due to the weak signal obtained from the small mitochondrial genome (~17 kb); inhibiting nuclear DNA replication does not increase sensitivity.
New Technique
The easy-to-use Click-iT® EdU assay, when combined with Tyramide Signal Amplification (TSA®) technology, enables sensitive and reliable measurement of nascent mtDNA synthesis. The Click-iT® EdU assay is much easier to perform, eliminating the use of radioactivity and avoiding the harsh DNA denaturation step required with the BrdU method, while TSA® technology has been reported to increase detection sensitivity up to 100-fold compared to conventional avidin–biotinylated enzyme complex (ABC) procedures.
TSA® is an enzyme-mediated detection method that uses the catalytic activity of horseradish peroxidase (HRP) to achieve high-density labeling of a target antigen. For the mtDNA detection assay, the target antigen is the fluorescent azide used in the Click-iT® EdU assay to detect DNA containing incorporated EdU. An HRP-conjugated anti-dye antibody is then combined with a tyramide analog that has the same fluorescence emission as the original fluorescent azide.
- Learn More About Click-iT® EdU
- Learn More About TSA® Technology and Kits
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Detecting mtDNA replication with TSA® and Click-iT® EdU in dissociated dorsal root ganglion neurons. Neurons plated on glass coverslips were incubated with 10 μM EdU. Following fixation and permeabilization, endogenous peroxidase activity was blocked and EdU was detected with Oregon Green® 488 azide. The Oregon Green® 488 signal was amplified using an HRP-conjugated rabbit antibody against Oregon Green® 488 and Alexa Fluor® 488 tyramide from TSA® Kit #12. The pan-neuronal marker αTuj1 was detected with a mouse primary antibody and visualized with an Alexa Fluor® 594 goat anti-mouse secondary antibody. Image contributed by Stephen I. Lentz and Eva L. Feldman, Departments of Internal Medicine and Neurology, University of Michigan. |
| Product | Quantity | Cat. No. | |
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| EdU (5-ethynyl-2'-deoxyuridine) | 50 mg | A10044 |  |
| Click-iT® Cell Reaction Buffer Kit | 1 kit | C10269 | |
| Oregon Green® 488 azide (Oregon Green® 6-carboxamido-(6-azidohexanyl), triethylammonium salt) *6-isomer* | 0.5 mg | O10180 | |
| Anti-fluorescein/Oregon Green®, rabbit IgG fraction, horseradish peroxidase conjugate | 0.5 mg | A21253 | |
| TSA® Kit #12 *with HRP–goat anti-rabbit IgG and Alexa Fluor® 488 tyramide* *50-150 slides* | 1 kit | T20922 | |
| Alexa Fluor® 594 goat anti-mouse IgG (H+L) *highly cross-adsorbed* *2 mg/mL* | 0.5 mL | A11032 | |
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