ProbesOnline October 2009

In This Issue


The Next Generation of AntibodiesABfinity™ Recombinant Monoclonal Antibodies

Illuminate Internalization Pathways in Real Time pHrodo™ Dextran for Endocytosis

Advancing Live-Cell Imaging Applications BacMam Enhancer Kit

New Products for Cell &Tissue Analysis
See all of this month's New Products for Cell & Tissue Analysis


Check out the latest issue of BioProbes

BioProbes 60   COVER STORY:
Neurons in Focus NEW!

Also download the
Neural stem cell reagent poster!

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what it is
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.

 ABfinity™ rabbit momoclonal antibody

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 Order Now
Rab11 100 μg 700184 Hu (X, Rt, P, Ms, Eq, Ch, B) WB, IF/ICC Order Now
Mnk1 [pT197/pT202] 100 μg 700242 Hu (Z, X, Sw, Rt, P, Ms, Mk (Rh), Eq, Cp, Ch, Cn, B) WB, F, IF/ICC Order Now
Cul-2 100 μg 700179 Rt, Ms, Hu (X, Or, Mk (Rh), Eq, Cp, Cn, B) WB, F, IHC, IF/ICC Order Now
AF-6 100 μg 700193 Ms, Hu (Mk, Cp, Cn, B, Rt) WB, IHC Order Now
IRAK4 100 μg 700026 Hu (Sw, Sh, Rt, Qu, Eq, Cn, B) F, IF/ICC Order Now
T-bet 100 μg 700059 Hu (Cp) WB, F, IHC, IF/ICC Order Now
PA28γ 100 μg 700180 Ms, Hu, Rt (Z, X, Or, Eq, Ch, Cn) WB, F, IHC, IF/ICC Order Now
AMPKβ1 [pS182] 100 μg 700241 Hu (X, Rt, Or, Eq, Ch, Cn, B, Ms) WB, F, IHC, IF/ICC Order Now
Pyk2 100 μg 700183 Hu (Rt, Or, Ms, Mk, Eq, Cp, Cn, B) F, IHC, IF/ICC Order Now
Smad1/5 [pS463/pS465] 100 μg 700047 Hu (Z, X, Sw, Sh, Su, Rt, Ms, Eq, Cp, Ch, Cn, B) F, IHC, IF/ICC Order Now
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
F, flow cytometry; ICC, immunocytochemistry; IF, immunofluorescence; IHC, immunohistochemistry; IP, immunoprecipitation; WB, western blotting

what it is
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
how it works
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.

pHrodo Dextran

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.
Product Quantity Cat. No.  
pHrodo™ dextran for endocytosis 0.5 mg P10361 Order Now
Organelle Lights™ Endosome-GFP 1 kit O10104 Order Now
Alexa Fluor® 647 transferrin 5 mg T23366 Order Now
what it is
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
how it works
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.

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.

Product Quantity Cat. No.  
BacMam Enhancer Kit
1 kit B10107 Order Now


The Click-iT® EdU cell proliferation assay is a superior alternative to traditional methods for detecting and quantitating newly synthesized DNA. Click-iT® assays use a modified nucleoside, EdU (5-ethynyl-2’-deoxyuridine), which is incorporated into DNA during active DNA synthesis. Detection of EdU is based on a click reaction, which is a copper (I)-catalyzed reaction between an azide and an alkyne. The EdU contains the alkyne, which can be reacted with an azide-containing detection reagent, to form a very stable triazole ring. Click-iT® EdU eliminates harsh treatments required by antibody-based methods, i.e, BrdU, providing a method that is easier and more reliable.

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.

DNA Synthesis

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

Species   Reference
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

Product Quantity Cat. No.  
Click-iT® AHA 5 mg C10102 Order Now
Click-iT® Tetramethylrhodamine Protein Analysis Detection kit 1 kit C33370 Order Now
Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay 1 kit C10289 Order Now
Click-iT® L-homopropargylglycine (HPG) 5 mg C10186 Order Now
Click-iT® Protein Reaction Buffer Kit 1 kit C10276 Order Now
tetramethylrhodamine (TAMRA) azide (tetramethylrhodamine 5-carboxamido-(6-azidohexanyl)) 0.5 mg T10182 Order Now
tetramethylrhodamine (TAMRA) alkyne (5-carboxytetramethylrhodamine, propargylamide) 0.5 mg T10183 Order Now
Free Online Technical Webinars 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.
Webinars Date Time
Mitochondrial Biology October 27, 2009 10:00 a.m. PT
Tools for iPSC Research November 10, 2009 10:00 a.m. PT

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.

Quantity Cat. No.
Alexa Fluor® 488 phalloidin
300 units
A12379 Order Now
4´,6-diamidino-2-phenylindole, dihydrochloride (DAPI) 10 mg
D1306 Order Now
Alexa Fluor® 568 goat anti-mouse IgG (H+L) 0.5 mL A11004 Order Now
 Nuclear yellow (Hoechst S769121, trihydrochloride, trihydrate) 10 mg
N21485 Order Now

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.


Product Quantity Cat. No.  
Alexa Fluor® 350 carboxylic acid, succinimidyl ester 5 mg A10168 Order Now
Alexa Fluor® 405 carboxylic acid, succinimidyl ester 1 mg A30000 Order Now
Alexa Fluor® 488 carboxylic acid, succinimidyl ester 1 mg A20000 Order Now
Alexa Fluor® 532 carboxylic acid, succinimidyl ester 1 mg A20001 Order Now
Alexa Fluor® 546 carboxylic acid, succinimidyl ester 1 mg A20002 Order Now
Alexa Fluor® 555 carboxylic acid, succinimidyl ester 1 mg A20009 Order Now
Alexa Fluor® 568 carboxylic acid, succinimidyl ester 1 mg A20003 Order Now
Alexa Fluor® 594 carboxylic acid, succinimidyl ester 1 mg A20004 Order Now
Alexa Fluor® 633 carboxylic acid, succinimidyl ester 1 mg A20005 Order Now
Alexa Fluor® 647 carboxylic acid, succinimidyl ester 1 mg A20006 Order Now
Alexa Fluor® 660 carboxylic acid, succinimidyl ester 1 mg A20007 Order Now
Alexa Fluor® 680 carboxylic acid, succinimidyl ester 1 mg A20008 Order Now
Alexa Fluor® 700 carboxylic acid, succinimidyl ester 1 mg A20010 Order Now
Alexa Fluor® 750 carboxylic acid, succinimidyl ester 1 mg A20011 Order Now
Alexa Fluor® 790 carboxylic acid, succinimidyl ester 0.1 mg A30051 Order Now
Cell & Tissue Analysis Scientific Posters 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.


Molecular Probes® The Handbook

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