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In This Issue
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DEPARTMENTS
Check out the latest issue of BioProbes
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FEATURED NEW PRODUCTS
Copper-Free Protein Detection in Live Cells — Click-iT® DIBO Reagents
| 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 the labeling of azide-modified macromolecules without the 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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Light Up Mitochondria — CellLight® Mitochondria Reagents
| what they are CellLight® reagents are fluorescent protein–signal peptide fusions that permit accurate and specific targeting to cellular structures, including mitochondria, for live-cell imaging or fixed-cell analyses. It’s now easier than ever to visualize mitochondria in combination with other probes, such as the rhod calcium indicators. what they offer
| how they work Cellular labeling with CellLight® reagents employs BacMam technology, which uses a modified insect cell baculovirus coupled with a mammalian promoter as a vehicle to efficiently deliver and express genes in mammalian 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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Measure Acute-Phase Proteins — Serum Amyloid A (SAA) Mouse or Multispecies ELISA Kits
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NEW APPLICATIONS
Reveal Elusive Nucleosome Kinetics Using Click Chemistry
| A recent publication in Science (Science (2010) 328:1161) describes a new technique, “CATCH-IT”, that enables researchers to label and capture sites of histone replacement, which in turn can reveal genome-wide kinetics of nucleosomes. Briefly, cells are fed the methionine analog L-azidohomoalanine (Click-iT® AHA), which incorporates into proteins during active protein synthesis. Click-iT® AHA contains a very small modification: an azido moiety, which enables detection of newly synthesized proteins through a “click chemistry” reaction—formation of a copper-catalyzed triazole from the azide and a biotin alkyne. Following the click reaction and isolation of nuclei, histone proteins containing the biotin tag are affinity-purified using streptavidin-based magnetic beads. The universal histone replacement variant H3.3 is then used to measure nucleosome turnover. Click chemistry describes an extremely powerful class of reactions that occur between biologically unique moieties (e.g., an azide and an alkyne). Click reactions have several benefits: the reaction between the detection moieties is efficient; no extreme temperatures or solvents are required; the reaction product is stable; and the components of the reaction are bioinert, which means that no side reactions occur. This final point is the greatest advantage of this powerful technique; click chemistry–labeled molecules can be applied to complex biological samples and detected with high sensitivity.
| (A) Structures of methionine and Click-iT® AHA. (B) Click-iT® azide/alkyne reaction. |
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DEPARTMENTS
Buzzworthy
Mitochondria Supply Membranes for Autophagosome Biogenesis During Starvation
Hailey DW, Rambold AS, Satpute-Krishnan P et al. (2010) Cell 141(4):656–667.
Autophagy is the recycling of cytosolic and organelle components during times of starvation, a cellular function that is critical to survival. The process begins with the formation of the autophagosome—a multilamellar body that has captured a volume of cytosol. This structure then fuses with a lysosome to degrade the contents, which are then transported back into the cytosol for use by the cell. To clarify conflicting reports regarding the origins of the autophagosome, Hailey and colleagues observed its formation using a series of organelle-specific, fusion protein–expressing constructs and organelle-staining probes (LysoTracker® DND99 and MitoTracker® Red CMXRos). The team was able to determine that starvation-induced autophagosomes emerge from mitochondrial outer membranes. They further concluded that starvation-induced autophagosomes are distinct from those formed in response to ER stress or calcium perturbation, and that they do not arise as a result of mitophagy.
- View the bibliography reference
- Learn More about Probes for Organelles
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) | Visualizing histones, actin, and peroxisomes in fibroblast cells. This Indian muntjac deer epidermis fibroblast cell was labeled with histone H1 Alexa Fluor® 488 conjugate, Alexa Fluor® 350 phalloidin, and the SelectFX® Alexa Fluor® 488 Peroxisome Labeling Kit. Image contributed by Michael W. Davidson, National High Magnetic Field Laboratory, The Florida State University. |
Tools to Navigate Intracellular Calcium Signaling
Increases in intracellular calcium (Ca2+) control a diverse set of cellular processes. Even within a single cell, the magnitude and duration of a change in the level of Ca2+ can vary greatly. Observing these concentration changes within a cell requires a diverse portfolio of detection reagents. We offer a complete range of reagents for calcium imaging applications.
When choosing a dye for a particular imaging application, dye emission intensities are a key consideration. The green-fluorescent fluo family of dyes has a very low emission at rest, resulting in a large increase in emission intensity upon Ca2+ binding, but making it difficult to resolve the cell. Members of the Oregon Green® BAPTA dye family have a higher resting fluorescence and therefore can be used to locate cells and structures; however, the maximal change in emission intensity is lower upon Ca2+ binding.
Generally, upon low-intensity stimulation, cells recruit a small number of plasma membrane or intracellular release channels. High-affinity dyes such as fluo-4, fluo-3, Oregon Green® BAPTA-1, and Oregon Green® BAPTA-2 are best-suited for these kinds of applications. A response to medium-intensity stimulation of cells, where many plasma membrane or intracellular channels are recruited for prolonged periods of time, is best detected using medium-affinity dyes such as fluo-5F. For high-intensity stimulation, low-affinity dyes such as Oregon Green® BAPTA-5N and fluo-4FF are most useful.
Choose red Ca2+ dyes for imaging cells or tissues with high autofluorescence, for simultaneous imaging of a green fluorophore, or for studies of calcium signaling, where these dyes enable simultaneous imaging of Ca2+ and a GFP chimera. As with green-emitting dyes, we offer both high-affinity red-emitting dyes (rhod-3 and X-rhod-1) and low-affinity dyes such as X-rhod-5F.
- Learn More about Calcium Sensors
| Imaging Ca2+ signaling in HeLa cells. HeLa cells were loaded with 5 µM rhod-3 AM (red), followed by 5 µM mag-fluo-4 (green), both at room temperature. Cells were then stimulated with histamine (100 µM) to release Ca2+ from the ER (decrease in mag-fluo-4 trace, green) into the cytoplasm (increase in rhod-3 signal, red). |
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| Product | Quantity | Cat. No. | |
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| Oregon Green® BAPTA-1, AM | 10 x 50 µg | O6807 |  |
| Oregon Green® BAPTA-2, AM | 10 x 50 µg | O6809 | |
| Oregon Green® BAPTA-5N, AM | 10 x 50 µg | O6813 | |
| Fluo-4, AM | 10 x 50 µg | F14201 | |
| Fluo-3, AM | 10 x 50 µg | F1242 | |
| Fluo-5F, AM | 10 x 50 µg | F14222 | |
| Fluo-4 FF, AM | 10 x 50 µg | F23981 | |
| Mag-Fluo-4, AM | 10 x 50 µg | M14206 | |
| X-Rhod-1, AM | 10 x 50 µg | X14210 | |
| X-Rhod-5F, AM | 10 x 50 µg | X23985 | |
| Rhod-3 Imaging Kit | 10 x 50 µg | R10145 | |
Research Tools for Influenza Confirmation and Analysis
Life Technologies supplies the tools, reagents, and support necessary for whole-genome sequencing, resequencing, and antiviral resistance testing of influenza viruses. For antiviral resistance testing, the NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Kit includes everything you need to quantitate neuraminidase activity and neuraminidase inhibitor resistance of avian, equine, human, and porcine influenza viruses. Check back for new kits available in September!
- Learn More about Applied Biosystems® Influenza Research Tools
Molecular Probes® The Handbook
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