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In This Issue
FEATURED NEW PRODUCTS
Imaging Reagents and Protocols at Your Fingertips — Cell Imaging App Now Available for the iPad®
| what it is
| how it works
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Quantitate Cell Viability at Your Benchtop — Tali™ Viability Kits
| what they are
| how they work
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| Product | Quantity | Cat. No. | |
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| Tali™ Image-Based Cytometer | 1 kit | T10796 |  |
| Tali™ Viability Kit—Dead Cell Red | 1 kit | A10786 | |
| Tali™ Viability Kit—Dead Cell Green | 1 kit | A10787 | |
Unravel the Molecular Mechanisms of Insulin Resistance — ABfinity™ Recombinant Rabbit Monoclonal Antibodies for AKT Substrates
| what they are
| how they work
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![Western blot analysis of whole cell extract using AS160 [pT642] ABfinity™ Recombinant Rabbit Monoclonal Antibody](javascript:CFC_popup('/etc/medialib/en/images/ics_organized/other/probesonline/0811.Par.43794.Image.-1.-1.1.gif','','resizable=yes,location=no,menubar=no,scrollbars=no,status=no,toolbar=no,fullscreen=no,dependent=no,width=491,height=640'))
| Product | Quantity | Cat. No. | |
|---|---|---|---|
| AS160 [pT642] ABfinity™ Recombinant Rabbit Monoclonal Antibody | 100 µg | 700565 | |
| AS160 [pT642] Recombinant Rabbit Oligoclonal Antibody | 100 µg | 710105 | |
| PRAS40 ABfinity™ Recombinant Rabbit Monoclonal Antibody | 100 µg | 701075 | |
| PRAS40 Recombinant Rabbit Oligoclonal Antibody | 100 µg | 710079 | |
| PRAS40 [pT246] Recombinant Rabbit Oligoclonal Antibody | 100 µg | 710089 | |
NEW APPLICATIONS
Spatiotemporal Imaging of Protein Synthesis and Autophagy
| The CellLight® reagents and Premo™ Autophagy Sensors combine the utility and selectivity of fluorescent proteins with the transduction efficiency of BacMam technology, enabling unambiguous visualization of organelles, cellular structures, and processes in live mammalian cells by fluorescence microscopy. Provided in ready-to-use format—simply add, incubate, and visualize—these reagents open new avenues for multiparametric studies of dynamic cellular events. Unlike expression vectors, BacMam reagents allow controlled and reproducible transient expression. CellLight® and Premo™ Autophagy BacMam 2.0 reagents provide several advantages over other gene transfer methods:
The combination of these reagents with more traditional imaging tools has been illustrated in several recent elegant studies. Narita et al. proposed a mechanism for the mTOR-dependent coregulation of protein synthesis and autophagic degradation during Ras-induced senescence. The authors revealed a self-enhancing system enabled by close spatial integration of rER-Golgi, autolysosomes, and mTOR, in which autolysosome-derived amino acids reinforce mTOR enrichment and activity. The authors suggested that this “TOR-autophagy spatial coupling compartment” (TASCC) may represent a general mechanism for rapid protein turnover. In addition to employing Premo™ Autophagy Sensor LC3B-RFP and CellLight® Golgi-GFP for colocalization studies, the authors used Click-iT® reagents based on “clickable” amino acid analogs (Click-iT® HPG and Click-iT® AHA) to visualize nascent proteins. |
- Learn More About Click-iT® Detection of Protein Synthesis
- Learn More About Premo™ Autophagy Sensor
- Learn More About CellLight® reagents
) | Visualization of nascent protein synthesis in Ras-induced senescent IMR90 cells. Cells were precultured for 30 min in methionine-free medium before metabolic labeling with a reactive methionine analog, Click-iT® L-homopropargylglycine (HPG). After a 30 min labeling with HPG, cells were fixed and stained for HPG and TGN46 (a marker of the trans-Golgi network, TGN). Nascent protein was accumulated in the TGN. Image used with permission from Masako Narita, Cambridge Research Institute. |
| Product | Quantity | Cat. No. | |
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| Premo™ Autophagy Sensor LC3B-RFP (BacMam 2.0) | 1 kit | P36236 | |
| Premo™ Autophagy Sensor LC3B-GFP (BacMam 2.0) | 1 kit | P36235 | |
| CellLight® Early Endosomes-RFP (BacMam 2.0) | 1 mL | C10587 | |
| CellLight® Golgi-GFP (BacMam 2.0) | 1 mL | C10592 | |
| Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay | 1 kit | C10289 | |
| Click-iT® AHA (L-Azidohomoalanine) | 5 mg | C10102 | |
| Click-iT® L-Homopropargylglycine (HPG) | 5 mg | C10186 | |
| CellLight® Lysosomes-GFP (BacMam 2.0) | 1 mL | C10596 | |
| CellLight® Lysosomes-RFP (BacMam 2.0) | 1 mL | C10597 | |
| Click-iT® Cell Reaction Buffer | 1 kit | C10269 | |
PROVEN PERFORMERS
Copper-Free Click-iT® DIBO Reagents Preserve Cell Viability and Protein Function
Dibenzocyclooctyne (DIBO) compounds react with azide-modified macromolecules to form a stable, covalent 1,2,3-triazole linkage in the absence of copper, enabling the study of live cells without copper toxicity. The use of DIBO compounds also allows simultaneous imaging with fluorescent proteins, which can be quenched in the presence of high copper concentrations. Particularly suited for imaging applications are the Click-iT® DIBO Alexa Fluor® compounds. Additionally, a DIBO handle can be introduced into proteins and other macromolecules with reactive probes capable of modifying amines, thiols, and carboxy groups. The Click-iT® biotin DIBO derivative is a means of introducing biotin groups at any azide-modified position in a biological molecule, allowing detection by microscopy and flow cytometry, and enrichment by techniques involving streptavidin or avidin. |
- Learn More About Click Chemistry Tools for Biological Assays
) | Live-cell labeling of surface glycoproteins with Click-iT® Alexa Fluor® DIBO compounds. HeLa cells were metabolically labeled for 3 days with tetraacetylated N-azidoacetyl-D-mannosamine (ManNAz), which introduces the N-azidoacetyl analog of sialic acid (SiaNAz) into cell surface and secreted glycoproteins. Click-iT® DIBO-Alexa Fluor® 488, Click-iT® DIBO-Alexa Fluor® 555, Click-iT® DIBO-Alexa Fluor® 594, or Click-iT® DIBO-Alexa Fluor® 647 was used to detect the cell surface labeled sialoglycoproteins. The nuclei were visualized with Hoechst 33342. Cells were imaged after fixation with 4% formaldehyde. Images were collected with the Thermo Scientific Cellomics ArrayScan® VTI platform. Top panels: control cells not treated with ManNAz; bottom panels: cells treated with ManNAz. |
| Product | Quantity | Cat. No. | |
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| Click-iT® Alexa Fluor® 488 DIBO Alkyne, for copper-free click chemistry detection of azide | 500 µg | C10405 | |
| Click-iT® Alexa Fluor® 555 DIBO Alkyne, for copper-free click chemistry detection of azide | 500 µg | C10406 | |
| Click-iT® Alexa Fluor® 594 DIBO Alkyne, for copper-free click chemistry detection of azide | 500 µg | C10407 | |
| Click-iT® Alexa Fluor® 647 DIBO Alkyne, for copper-free click chemistry detection of azide | 500 µg | C10408 | |
| Click-iT® TAMRA DIBO Alkyne, for copper-free click chemistry detection of azide | 500 µg | C10410 | |
| Click-iT® Amine DIBO Alkyne, for copper-free click chemistry | 1 mg | C10411 | |
| Click-iT® Biotin DIBO Alkyne, for copper-free click chemistry | 1 mg | C10412 | |
| Click-iT® Maleimide DIBO Alkyne, for copper-free click chemistry | 1 mg | C10413 | |
| Click-iT® Succinimidyl Ester DIBO Alkyne, for copper-free click chemistry | 1 mg | C10414 | |
| Click-iT® ManNAz Metabolic Glycoprotein Labeling Reagent (tetraacetylated N-azidoacetyl-D-mannosamine) | 1 each | C33366 | |
DEPARTMENTS
From the Bench
Targeted Identification of Metastasis-Associated Cell-Surface Sialoglycoproteins in Prostate Cancer
Yang L, Nyalwidhe JO, Guo S, Drake RR, Semmes OJ (2011) Mol Cell Proteomics 10(6):M110.007294.
Yang et al. recently described a method using Click-iT® ManNAz metabolic glycoprotein labeling reagent (Ac4ManNAz) for the metabolic labeling, enrichment, and identification of sialoglycoproteins in nonmetastatic vs. metastatic syngenic prostate cancer cell lines. The cultured cells were fed Ac4ManNAz for 1–3 days, and to confirm metabolic labeling of cell-surface glycoproteins, the cells were fixed and click-labeled with the Click-iT® Biotin Protein Analysis Detection Kit and analyzed by confocal microscopy and flow cytometry. After confirmation of labeling, the azide-labeled cellular lysates were reacted with biotin-alkyne, and the labeled sialoglycoproteins were enriched on streptavidin resin. The resin-bound glycoprotein fraction was eluted and analyzed by SDS-PAGE followed by liquid chromatography–tandem MS. The results showed that the Click-iT® assay–based enrichment approach gave, on average, a 3-fold increase in the number of cell surface sialylated glycoproteins identified. The results also showed that multiple sialoglycoproteins are overexpressed in the metastatic cell lines and that most of these proteins are known to be involved in cell motility, migration, and invasion.
- View the Bibliography Reference
- Learn More about Click Chemistry Tools for Biological Assays
On the Web
 | Get a Free Sample of SlowFade® Gold or ProLong® Gold Our antifade kits and reagents have been shown to increase the photostability of, and to reduce initial quenching of, many of our fluorophores in fixed cells, fixed tissues, and cell-free preparations. Through December 31, 2011, get a free sample of SlowFade® Gold or ProLong® Gold antifade reagent.*
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Imaging Corner
) | Wound healing in neonatal dermal fibroblasts. (Left) 24 hours post-wound, treated with vehicle. (Right) 24 hours post-wound, treated with 0.1 µM cytochalasin D. Gibco® human neonatal dermal fibroblasts were stained with Alexa Fluor® 488 phalloidin, anti-tubulin IgG, and goat-anti mouse Alexa Fluor® 555 conjugate.
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