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In This Issue
The Next Generation of Antibodies — ABfinity™ Recombinant Monoclonal Antibodies
Illuminate Internalization Pathways in Real Time — pHrodo™ Dextran for Endocytosis
Advancing Live-Cell Imaging Applications — BacMam Enhancer Kit
See all of this month's New Products for Cell & Tissue Analysis
- Buzzworthy — Visualizing Axonal Response to Growth and Guidance Factors
- Online Technical Webinars — Mitochondrial Biology & iPSC Research
- The View — Staining so Bright, it's Scary
- Proven Performers — Amine-reactive Alexa Fluor® Dyes
- On the Web — Cell & Tissue Analysis Scientific Posters & Secondary Antibody Selection Tool
Check out the latest issue of BioProbes
Neurons in Focus NEW!
Also download the
Neural stem cell reagent poster!
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FEATURED NEW PRODUCTS
ABfinity™ antibodies are the next generation of antibodies, presented exclusively by Invitrogen. These antibodies are generated by cloning the specific antibody genes and producing them in a mammalian expression system. With ABfinity™ technology, we have generated the most specific antibodies available, bringing you highly reproducible results.
what it offers
- Unmatched lot-to-lot consistency
- High sensitivity and specificity
- Extensive validation and characterization
how it works
Generating antibodies by expressing cloned antibody genes ensures that every lot of ABfinity™ antibodies produces the same results. These antibodies offer maximal sensitivity and specificity, allowing you to detect small amounts of only your intended targets. ABfinity™ antibodies are also extensively validated and characterized, giving you the confidence you need, right out of the tube—no further optimization is required.
HeLa cells labeled with ABfinity™ rabbit monoclonal antibody against the eukaryotic initiation factor 4EBP1 [pT37] in the absence of peptides (left), and in the presence of a phosphopeptide (center) or nonphosphopeptide (right) used as an immunogen. Detection was performed with Alexa Fluor® 488 goat anti-rabbit IgG. Actin was stained with Alexa Fluor® 568 phalloidin.
ABfinity™ Rabbit Monoclonal Antibodies
||Quantity||Cat. No.||Reactivity: validated (expected)||Applications||
|PKC-θ [pT538]||100 μg||700043||Hu (X, Rt, Ms, Cp, B)||WB, F, IHC, IF/ICC|
|Rab11||100 μg||700184||Hu (X, Rt, P, Ms, Eq, Ch, B)||WB, IF/ICC|
|Mnk1 [pT197/pT202]||100 μg||700242||Hu (Z, X, Sw, Rt, P, Ms, Mk (Rh), Eq, Cp, Ch, Cn, B)||WB, F, IF/ICC|
|Cul-2||100 μg||700179||Rt, Ms, Hu (X, Or, Mk (Rh), Eq, Cp, Cn, B)||WB, F, IHC, IF/ICC|
|AF-6||100 μg||700193||Ms, Hu (Mk, Cp, Cn, B, Rt)||WB, IHC|
|IRAK4||100 μg||700026||Hu (Sw, Sh, Rt, Qu, Eq, Cn, B)||F, IF/ICC|
|T-bet||100 μg||700059||Hu (Cp)||WB, F, IHC, IF/ICC|
|PA28γ||100 μg||700180||Ms, Hu, Rt (Z, X, Or, Eq, Ch, Cn)||WB, F, IHC, IF/ICC|
|AMPKβ1 [pS182]||100 μg||700241||Hu (X, Rt, Or, Eq, Ch, Cn, B, Ms)||WB, F, IHC, IF/ICC|
|Pyk2||100 μg||700183||Hu (Rt, Or, Ms, Mk, Eq, Cp, Cn, B)||F, IHC, IF/ICC|
|Smad1/5 [pS463/pS465]||100 μg||700047||Hu (Z, X, Sw, Sh, Su, Rt, Ms, Eq, Cp, Ch, Cn, B)||F, IHC, IF/ICC|
|Reactivity: B, bovine; Ch, chicken; Cn, canine; Cp, chimpanzee; Eq, equine; Fe, feline; Gf, goldfish; Ha, hamster; Hu, human; Ma, mammalian; Mk, monkey (rhesus); Ms, mouse; Ne, nematode; Or, orangutan; P, primate; Qu, quail; Rb, rabbit; Rt, rat; Sh, sheep; Su, sea urchin; Sw, swine; X, Xenopus; Z, zebrafish
Applications: F, flow cytometry; ICC, immunocytochemistry; IF, immunofluorescence; IHC, immunohistochemistry; IP, immunoprecipitation; WB, western blotting
pHrodo™ dextran is a superior alternative to other fluorescent dextran conjugates (e.g., BCECF and tetramethylrhodamine [TRITC]) for live-cell imaging of endocytosis.
what it offers
- Content-rich results—orange-red fluorescence facilitates multiplexing with blue, green, and far-red fluorescent fluorophores
- Simple method—minimal fluorescent signal at neutral pH eliminates quenching reagents and extra wash
pHrodo™ dextran has a pH-sensitive fluorescence emission (excitation and emission maxima of 560/585 nm) that increases in intensity with increasing acidity. This increase is particularly dramatic in the pH range of 4 to 8, commonly seen as endocytic vesicles are acidified. pHrodo™ dextran is essentially dark in the extracellular environment. Upon internalization, the acidic environment of the endosome elicits a bright, red-fluorescent signal that can be visualized by fluorescence microscopy, flow cytometry, or high-content imaging and analysis.
Multiplexed analysis with pHrodo™ dextran for endocytosis. HeLa cells were transduced with Organelle Lights™ Endosome-GFP. The following day, medium was replaced with serum-free medium plus 50 µM deferoxamine. Cells were washed and incubated with 1 µg/mL Hoechst 33342, 10 µg/mL pHrodo™ Dextran, and Alexa Fluor® 647 transferrin for 5 min at 37°C. (A) Organelle Lights™ Endosome-GFP; (B) Alexa Fluor® 647 transferrin; (C) pHrodo™ dextran.
|pHrodo™ dextran for endocytosis||0.5 mg||P10361|
|Organelle Lights™ Endosome-GFP||1 kit||O10104|
|Alexa Fluor® 647 transferrin||5 mg||T23366|
The BacMam Enhancer Kit provides researchers using BacMam reagents with additional BacMam enhancer. Each kit provides sufficient material for treating 100 coverslips or 10 x 96-well microplates.
what it offers
- Convenience—packaging and protocol is identical to the enhancer provided in Cellular Lights™, Organelle Lights™, and Premo™ biosensor kits
- Productivity—increased reagent expression in mammalian cells
- Stability—enhancer solution withstands multiple freeze/thaw cycles
The BacMam enhancer helps to increase expression of fluorescent protein–signal peptide fusions following transduction by baculovirus-based BacMam reagents. BacMam reagents include the Premo™ biosensors for chloride, sodium, and the cell cycle, as well as Organelle Lights™ and Cellular Lights™ fluorescent proteins. Each of these products includes BacMam enhancer as a component; however, additional BacMam enhancer may be required for some cell types or applications. Provided in ready-to-use format, BacMam reagents open up new avenues for multiparametric studies of dynamic cellular events in live cells.
- Learn More about BacMam Technology
Live-cell visualization of cytoskeletal and mitochondrial dynamics and organization with BacMam technology. HeLa cells were incubated with Cellular Lights™ Talin-RFP and Organelle Lights™ Mito-GFP for ~2 hr, followed by treatment with BacMam enhancer. Cells were washed and incubated overnight to allow protein expression. Imaging was performed on live cells using a DeltaVision® Core microscope and standard DAPI/FITC/TRITC filter sets.
Although the Click-iT® EdU assay has only been available since 2007, our data and several publications already demonstrate its use in a wide variety of species—covering plants, bacteria, yeast, and a broad spectrum of animals including flatworm, zebrafish, mouse, rat, and human. Even with plant cells, Click-iT® EdU assays involve only a mild fixation and permeabilization step—no DNA denaturation or cell wall digestion is required.
- Learn More about Click-iT® EdU Cell Proliferation Assays
Detection of DNA synthesis in Flagellophora cf. apelti. Cells were exposed to the nucleoside analog EdU (100 µM in sea water) for 10 hr. Following fixation and permeabilization, EdU that had been incorporated into newly synthesized DNA was detected with the Click-iT® EdU Alexa Fluor® 488 Imaging Kit (green fluorescence). Phospho-H3 was detected using a rabbit primary antibody followed by an Alexa Fluor® 568 dye–labeled goat anti–rabbit IgG antibody (red fluorescence). Image submitted by Julian Smith, Department of Biology, Winthrop University, USA.
References: Click-iT® EdU in Various Organisms
|Bacteria||Ferullo D et al. (2009) Methods 48:8–13|
|Flatworm (marine)||See Figure for experimental details.|
|Zebrafish larva||BioProbes 57|
|Zebra Finch||Scientific poster, ASCB 2007*|
|Mouse||Salic A (2008) Proc Natl Acad Sci U S A 105:2415–2420|
|Bonaguidi M et al. (2008) J Neurosci 28:9194–9204|
|Kharas MG et al. (2008) J Clin Invest 118:3038–3050|
|Plants||Vanstraelen M et al. (2009) Proc Natl Acad Sci U S A 106:11806–11811|
|Rat||Scientific poster, ASCB 2007*|
|Human||McCord A et al. (2009) Clin Cancer Res 15:5145–5153|
|Momcilovic O et al. (2009) Stem Cells 27:1822–1835|
|* Download the scientific posters|
Using Click-iT® Reagents to Visualize Axonal Response to Growth and Guidance Factors
Protein synthesis in distal axons is not required for growth cone responses to guidance cues.
Roche FK et al. (2009) J Neurosci 29:638–652.
Are proteins made in the axon required for axon growth and guidance?
Neuronal growth cones are dynamic regions of developing neurons that respond to external stimuli in order to help axons find their target synapse. Although it is known that proteins produced by the axon have important neurobiological roles, the roles these locally synthesized proteins may play in axonal motility and navigation is unclear.
Using Click-iT® reagents to visualize nascent protein synthesis, Roche and colleagues examined the growth cone responses of chick and mouse neurons to various axonal guidance cues, in the local or global presence of protein synthesis inhibitors.
Growth cone response to NGF diminished after 1 hr of global inhibition of protein synthesis. However, by using compartmented growth dishes that allowed selective treatment of different neuronal regions, they observed that suppressing protein synthesis in the axon only allowed undiminished elongation of axons to continue for 24 hr or more.
These results suggest that protein synthesis in neuronal regions distal to the growth cone is sufficient for supporting axonal elongation and growth cone response.
View bibliography reference
|Click-iT® AHA||5 mg||C10102|
|Click-iT® Tetramethylrhodamine Protein Analysis Detection kit||1 kit||C33370|
|Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay||1 kit||C10289|
|Click-iT® L-homopropargylglycine (HPG)||5 mg||C10186|
|Click-iT® Protein Reaction Buffer Kit||1 kit||C10276|
|tetramethylrhodamine (TAMRA) azide (tetramethylrhodamine 5-carboxamido-(6-azidohexanyl))||0.5 mg||T10182|
|tetramethylrhodamine (TAMRA) alkyne (5-carboxytetramethylrhodamine, propargylamide)||0.5 mg||T10183|
|Free online technical webinars
You are invited to join us for a series of biweekly technical webinars from the comfort of your desk. The webinars will initially focus on imaging-related applications, but we welcome your feedback for additional topics throughout the course of the year. Upcoming topics will be announced each month via email.
Presentations will last approximately 45 minutes, followed by 15 minutes for live Q&A.
Missed our previous webinars? Find our recorded webinars here!
Staining so bright, it's scary. (Left) Formaldehyde-fixed and permeabilized 3T3 mouse fibroblast cell labeled with Alexa Fluor® 488 phalloidin (green, F-actin) and DAPI (blue, nuclei), then mounted in ProLong® antifade mounting medium. (Right) Formaldehyde-fixed and permeabilized BPAE cells labeled with mouse anti–alpha-tubulin primary antibody and Alexa Fluor® 568 goat anti-mouse secondary antibody. Nuclei were labeled using Nuclear Yellow. Cells were mounted in ProLong® antifade mounting medium.
|Alexa Fluor® 488 phalloidin
|4´,6-diamidino-2-phenylindole, dihydrochloride (DAPI)||10 mg
|Alexa Fluor® 568 goat anti-mouse IgG (H+L)||0.5 mL||A11004|
|Nuclear yellow (Hoechst S769121, trihydrochloride, trihydrate)||10 mg
Proven Performers — Amine-reactive Alexa Fluor® Dyes
In addition to offering expertly prepared dye conjugates, we also provide you with the opportunity to create your own fluorescent conjugates using reactive dyes. The Alexa Fluor® dyes—a series of superior fluorescent dyes that span the near-UV, visible, and near-IR spectrum—produce the best and brightest conjugates.
For selectively linking an Alexa Fluor® dye to accessible primary amine groups on proteins, modified nucleic acids, or other molecules, Alexa Fluor® succinimidyl esters provide the easiest and most efficient reaction chemistry. Succinimidyl esters are excellent reagents for amine modification because the covalent bonds they form are as stable as the peptide bonds used to link amino acids in proteins.
With these reagents, you can vary both the amount of dye and the target in your labeling reaction to create the perfect Alexa Fluor® conjugate for your research application. These reactive dyes are also used in our Protein Labeling Kits, Monoclonal Antibody Labeling Kits, APEX™ Labeling Kits, and Oligonucleotide Labeling Kits.
Two-color confocal image of a human epidermal whole mount. b1 integrin was visualized with the monoclonal antibody P5D2 labeled with the green-fluorescent Alexa Fluor® 488 dye using the Alexa Fluor® 488 Monoclonal Antibody Labeling Kit. a6 integrin was labeled using a mouse monoclonal antibody visualized with the Alexa Fluor® 594 goat anti–mouse IgG antibody and pseudocolored blue. Image contributed by Uffe Birk Jensen, Department of Human Genetics, University of Aarhus, Denmark.
|Alexa Fluor® 350 carboxylic acid, succinimidyl ester||5 mg||A10168|
|Alexa Fluor® 405 carboxylic acid, succinimidyl ester||1 mg||A30000|
|Alexa Fluor® 488 carboxylic acid, succinimidyl ester||1 mg||A20000|
|Alexa Fluor® 532 carboxylic acid, succinimidyl ester||1 mg||A20001|
|Alexa Fluor® 546 carboxylic acid, succinimidyl ester||1 mg||A20002|
|Alexa Fluor® 555 carboxylic acid, succinimidyl ester||1 mg||A20009|
|Alexa Fluor® 568 carboxylic acid, succinimidyl ester||1 mg||A20003|
|Alexa Fluor® 594 carboxylic acid, succinimidyl ester||1 mg||A20004|
|Alexa Fluor® 633 carboxylic acid, succinimidyl ester||1 mg||A20005|
|Alexa Fluor® 647 carboxylic acid, succinimidyl ester||1 mg||A20006|
|Alexa Fluor® 660 carboxylic acid, succinimidyl ester||1 mg||A20007|
|Alexa Fluor® 680 carboxylic acid, succinimidyl ester||1 mg||A20008|
|Alexa Fluor® 700 carboxylic acid, succinimidyl ester||1 mg||A20010|
|Alexa Fluor® 750 carboxylic acid, succinimidyl ester||1 mg||A20011|
|Alexa Fluor® 790 carboxylic acid, succinimidyl ester||0.1 mg||A30051|
|New—Cell & Tissue Analysis Scientific Posters
Want to see our products in action?
Check out the new scientific posters web page in our Cell & Tissue Analysis application area. You’ll find posters presented at scientific meetings covering a broad range of platforms and applications—from flow cytometry to fluorescence microscopy, from cell physiology to apoptosis. You can also find our cell signaling pathway posters including the new kinome map.
|New and Improved—Secondary Antibody Selection Tool
Finding secondary antibodies just got easier than ever. We have redesigned the search tool to allow you to get to the antibody you need, faster and easier. This new format eliminates the need to scroll through long lists of products, and we have added the ability to search within your results. Try it today.
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