ProbesOnline October 2009

In This Issue

FEATURED NEW PRODUCTS

 
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


NEW APPLICATIONS

 
Detect Active DNA Synthesis in Any Organism
DEPARTMENTS



Check out the latest issue of BioProbes

BioProbes 60  COVER STORY:
Neurons in Focus NEW!

Also download the
Neural stem cell reagent poster!


Subscribe to BioProbes

 

FEATURED NEW PRODUCTS

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

Target
Quantity Cat. No. Reactivity: validated (expected) Applications
PKC-θ [pT538]100 μg700043Hu (X, Rt, Ms, Cp, B)WB, F, IHC, IF/ICCOrder Now
Rab11100 μg700184Hu (X, Rt, P, Ms, Eq, Ch, B)WB, IF/ICCOrder Now
Mnk1 [pT197/pT202] 100 μg700242Hu (Z, X, Sw, Rt, P, Ms, Mk (Rh), Eq, Cp, Ch, Cn, B)WB, F, IF/ICCOrder Now
Cul-2100 μg700179Rt, Ms, Hu (X, Or, Mk (Rh), Eq, Cp, Cn, B)WB, F, IHC, IF/ICCOrder Now
AF-6100 μg700193Ms, Hu (Mk, Cp, Cn, B, Rt)WB, IHCOrder Now
IRAK4100 μg700026Hu (Sw, Sh, Rt, Qu, Eq, Cn, B)F, IF/ICCOrder Now
T-bet 100 μg700059Hu (Cp)WB, F, IHC, IF/ICCOrder Now
PA28γ 100 μg700180Ms, Hu, Rt (Z, X, Or, Eq, Ch, Cn)WB, F, IHC, IF/ICCOrder Now
AMPKβ1 [pS182] 100 μg700241Hu (X, Rt, Or, Eq, Ch, Cn, B, Ms)WB, F, IHC, IF/ICCOrder Now
Pyk2 100 μg700183Hu (Rt, Or, Ms, Mk, Eq, Cp, Cn, B)F, IHC, IF/ICCOrder Now
Smad1/5 [pS463/pS465]100 μg700047Hu (Z, X, Sw, Sh, Su, Rt, Ms, Eq, Cp, Ch, Cn, B)F, IHC, IF/ICCOrder 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
Applications:
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 endocytosis0.5 mgP10361Order Now
Organelle Lights™ Endosome-GFP1 kitO10104Order Now
Alexa Fluor® 647 transferrin5 mgT23366Order 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 kitB10107Order Now

NEW APPLICATIONS

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

DEPARTMENTS

Buzzworthy

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.

Methods
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.

Results
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.

Conclusion
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® AHA5 mgC10102Order Now
Click-iT® Tetramethylrhodamine Protein Analysis Detection kit1 kitC33370Order Now
Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay1 kitC10289Order Now
Click-iT® L-homopropargylglycine (HPG)5 mgC10186Order Now
Click-iT® Protein Reaction Buffer Kit1 kitC10276Order Now
tetramethylrhodamine (TAMRA) azide (tetramethylrhodamine 5-carboxamido-(6-azidohexanyl))0.5 mgT10182Order Now
tetramethylrhodamine (TAMRA) alkyne (5-carboxytetramethylrhodamine, propargylamide) 0.5 mgT10183Order 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.



Product
Quantity Cat. No.
Alexa Fluor® 488 phalloidin
300 units
A12379Order Now
4´,6-diamidino-2-phenylindole, dihydrochloride (DAPI)10 mg
D1306Order Now
Alexa Fluor® 568 goat anti-mouse IgG (H+L)0.5 mLA11004Order Now
 Nuclear yellow (Hoechst S769121, trihydrochloride, trihydrate)10 mg
N21485Order 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 ester5 mgA10168Order Now
Alexa Fluor® 405 carboxylic acid, succinimidyl ester1 mgA30000Order Now
Alexa Fluor® 488 carboxylic acid, succinimidyl ester1 mgA20000Order Now
Alexa Fluor® 532 carboxylic acid, succinimidyl ester1 mgA20001Order Now
Alexa Fluor® 546 carboxylic acid, succinimidyl ester1 mgA20002Order Now
Alexa Fluor® 555 carboxylic acid, succinimidyl ester1 mgA20009Order Now
Alexa Fluor® 568 carboxylic acid, succinimidyl ester1 mgA20003Order Now
Alexa Fluor® 594 carboxylic acid, succinimidyl ester1 mgA20004Order Now
Alexa Fluor® 633 carboxylic acid, succinimidyl ester1 mgA20005Order Now
Alexa Fluor® 647 carboxylic acid, succinimidyl ester1 mgA20006Order Now
Alexa Fluor® 660 carboxylic acid, succinimidyl ester1 mgA20007Order Now
Alexa Fluor® 680 carboxylic acid, succinimidyl ester1 mgA20008Order Now
Alexa Fluor® 700 carboxylic acid, succinimidyl ester1 mgA20010Order Now
Alexa Fluor® 750 carboxylic acid, succinimidyl ester1 mgA20011Order Now
Alexa Fluor® 790 carboxylic acid, succinimidyl ester0.1 mgA30051Order 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.

 



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