Create your own bright label for imaging with the Qdot® 625 Antibody Conjugation Kit
 
what it is
Each Qdot® Antibody Conjugation Kit contains sufficient reagents, ultrafiltration columns, separation media, and associated components to perform two separate conjugation reactions. The protocol accompanying the kit details the method for conjugating Qdot® 625 nanocrystals to a 300 µg antibody sample, but the protocol can be adapted for conjugation to polyclonal Fab fragments and other classes of whole antibodies. Labeled antibodies can then be used in immunostaining (see figure) or other experiments requiring biofunctionalized labels (e.g., flow cytometry, ELISA).



Fixed and permeabilized HeLa cells were labeled with mouse anti–α-tubulin primary antibody and visualized with 20 nM Qdot® 625 goat anti–mouse IgG to show the microtubule network.


how it works

The Qdot® 625 Antibody Conjugation Kit (containing amine-derivatized, PEG-coated Qdot® nanocrystals and the amine–thiol cross linker SMCC) allows you to conjugate your own antibodies to our brightest visible dot with the peak of emission of 625 nm (see figure). The conjugation reaction can be completed in a few hours and is based on the fast and efficient coupling of thiols to reactive maleimide groups, which are present on the nanocrystals after SMCC activation. In addition to antibodies, other thiol-containing molecules can be coupled to the 625 nm nanocrystals using this kit.




The Qdot® 625 nanocrystal is characterized by a broad absorption spectrum and a narrow, symmetrical emission profile.

what it offers
  • the ultimate in fluorescence photostability and brightness
  • ease of use
  • flexibility to conjugate to most antibodies
  • compatibility with other Qdot® colors used with single-excitation sources for multispectral imaging with optimized filter sets

For more information about these and other Qdot® products, visit www.invitrogen.com/qdots. Also, be sure to investigate the BrightLine® QD625-A Filter Set—a new optimized filter set from Semrock, Inc. designed for use with Qdot® 625 nanocrystals.

Qdot® 625 Antibody Conjugation Kit
Measure JAK2, STAT3, and STAT5 phosphoproteins and more
 
what they are
Signal transducers and activators of transcription (STAT) are proteins that control the transcription of specific genes in response to cytokine stimulation. Perform quantitative analysis of phosphoproteins in the Jak/Stat pathway using our phosphoELISA™ kits or, for simple and efficient profiling, use the Luminex® Multiplex STAT1, 3, and 5a/b Phospho 3-Plex Antibody Bead Kit assays that require only 10,000 cells.

how they work
The JAK/STAT phosphoELISA™ kits provide single-protein measurements, while the STAT1, 3, 5a/b Phospho 3-Plex assay is designed to simultaneously quantify the levels of multiple phosphorylated proteins (see figure). Both phosphoELISA™ and Multiplex Luminex® assays are solid-phase sandwich immunoassays for measuring phosphorylated proteins in cellular extracts. In the phosphoELISA™ kits, the solid support is the microtiter plate (precoated with monoclonal antibodies), whereas in the Luminex® Multiplex Phospho 3-Plex kits, beads of defined spectral properties (conjugated to analyte-specific capture antibodies) serve as the solid support. For the Luminex® Multiplex assay, the conjugated beads and samples are added to the wells of a filter-bottom microplate and are incubated. During this first incubation, the target analyte (or analytes) is captured. After washing, a biotinylated anti-rabbit secondary antibody (with specificity to the target analyte) is added. After the secondary antibody is allowed to bind, detection is achieved by the addition of streptavidin R-PE (R-phycoerythrin). After washing to remove unbound anti–rabbit R-PE, the beads are analyzed with the Luminex® 100™ or 200™ instrument. 


STAT activation following treatment with IFN-α and IFN-γ. Phosphospecific multiplex bead immunoassay data illustrate patterns of STAT activation in each cell type treated with either IFN-α or IFN-γ.

what they offer

  • superior performance—accurate, reproducible, and sensitive quantitation of multiple proteins
  • high quality—in-house manufactured antibodies ensure excellent specificity and sensitivity
  • fast and easy protocols—perform and analyze your data in less than one day

Luminex® Multiplex Assays (100 tests) Cat. no.
STAT1, 3, 5a/b Phospho 3-Plex PanelLHO0005
STAT1 (Total) Antibody Bead KitLHO0261
STAT1 [pY701] Antibody Bead KitLHO0271
STAT3 [pY705] Antibody Bead KitLHO0481

phosphoELISA™ kits for JAK/STAT proteins (96 tests) Cat. no.
JAK2 (Total) phosphoELISA™ KitKHO5521
JAK2 [pYpY1007/1008] phosphoELISA™ KitKHO5621
STAT1 (Total) phosphoELISA™ KitKHO0261
STAT1 [pY701] phosphoELISA™ KitKHO0271
STAT3 (Total) phosphoELISA™ KitKHO0471
STAT3 [pY705] phosphoELISA™ KitKHO0481
STAT4 [pY1059] phosphoELISA™ KitKHO0621
STAT5a (Total) phosphoELISA™ KitKHO0751
STAT5a [pY694] phosphoELISA™ KitKHO0761
STAT5b [pY699] phosphoELISA™ KitKHO5721
STAT6 (Total) phosphoELISA™ KitKHO0791
STAT6 [pY641] phosphoELISA™ KitKHO0801
CellTracker™ Violet BMQC dye for cell tracking

what it is
CellTracker™ dyes are well retained in cells and are inherited by daughter cells in a population, making them ideal for cell lineage tracking. In addition to this new UV/405 nm–excitable dye, we also offer CellTracker™ dyes in blue, green, orange, and red fluorescent colors.

how it works
To load CellTracker™ dyes into cells, simply add the reagent to the culture medium, incubate, and wash prior to imaging. The CellTracker™ dye binds to intracellular thiols, which transforms it into a cell membrane–impermeant reaction product. CellTracker™ Violet BMQC is aldehyde fixable and can withstand permeabilization with Triton®X-100 (0.5%).

what it offers

  • ease of use
  • brighter than alternative products
  • consistent, repeatable results
  • excitation using UV or 405 nm light with emission monitored at 525 nm

CellTracker™ Violet BMQC
Internalization of β-amyloid peptide by primary neurons in the absence of apolipoprotein E.
Saavedra, L. et al. (2007) J Biol Chemistry 282: 35722.

Does intracellular β-amyloid peptide contribute to Alzheimer's disease? Extraneuronal accumulation of β-amyloid peptide (Aβ) is a central event in the pathogenesis of Alzheimer's disease (AD). However, little is known of the contribution of intracellular Aβ to AD or of the cellular mechanisms that govern its accumulation within neurons. Aβ in the brain and CSF is known to associate with apolipoprotein E (apoE), and internalization of the Aβ–apoE complex has been extensively studied. However, this complex exists in equilibrium with free Aβ the details of the neuronal uptake of this uncomplexed peptide are largely unknown. Saavedra and colleagues have investigated the uptake of uncomplexed Aβ using a culture system that allows selective treatment of axons and cell bodies. Their results demonstrate that Aβ is preferentially internalized via axons, where it is then transported intracellularly to cell bodies. A suggested possibility for this preferential uptake is the direct involvement of receptor proteins that are enriched in axons; however, further investigation ruled out a number of candidates for such a protein, including the nicotinic receptor α7nACHr and LDL receptors, as well as clathrin-dependent mechanisms. Using Alexa Fluor® dye detection of caveolin and cholera toxin B in endocytosis-impaired dynamin mutant cells, the authors showed that Aβ doesn't colocalize with caveolin, but partially colocalizes with cholera toxin B, suggesting a non-caveolae lipid raft route. Further, they showed that the internalization of free Aβ depends on cellular cholesterol and sphingolipid levels, in support of previous studies reporting that cholesterol is required for Aβ binding to the cell surface. However, their finding that functional dynamin is required for Aβ internalization reinforces the notion that the underlying uptake mechanism is endocytic. The authors propose that detailed understanding of the molecular mechanism underlying Aβ uptake may lead to the development of promising therapeutic strategies for AD.

View bibliography reference
To study the mutualism between vertebrates and their associated microbial communities, or microbiota, the Guillemin lab has developed a germ-free zebrafish model. Our analysis has revealed important roles for the gut microbiota in intestinal epithelial maturation, cell homeostasis and cell type specification, and the establishment of mucosal tolerance. We are currently investigating the molecular nature of the microbiota-derived signals that drive these developmental programs. One interesting phenotype we are exploring is the reduced rate of intestinal cell proliferation in zebrafish larvae reared without microbes. Typically, BrdU labeling is used to determine the number of proliferating cells in situ, but it is often not reliable and the harsh tissue treatment affects labeling with other antibodies.
 
In this image, a 6-day-old zebrafish larva was reared in a germ-free environment and then exposed to a 16 hour pulse of 100 μM EdU, a nucleoside analog which is incorporated into cells that are actively synthesizing DNA. The tissue was paraformaldehyde fixed, paraffin embedded, and cut in 7 micron sections. After deparaffinization, rehydration, permeabilization, and a 10 minute blocking step in 3% BSA + 0.5% Triton® X-100 in PBS, the tissue was labeled by the click reaction cocktail. The incorporated EdU was then labeled with the azide-modified click detection reagent for 30 minutes.
 
The image is a cross section through the mid intestine. Proliferating nuclei appear green due to labeling with Alexa Fluor® 488 azide from the Click-iT™ EdU Alexa Fluor® 488 Imaging Kit and are counterstained blue with DAPI to detect all nuclei in the tissue. The large circular void in the lower center of the image is the intestinal lumen; numerous actively proliferating intestinal cells can be seen in the surrounding epithelial wall in green. The monochromatic 20× image was captured using a CoolSNAP camera (Photometrics Systems) mounted on a Nikon Eclipse TE2000-V inverted microscope (200 msec exposure time). The image was processed (including pseudocoloring) using Photoshop® software (Adobe, Inc.). Image submitted by Sarah Cheesman, Institute of Molecular Biology, University of Oregon, USA.
 

New Jak/Stat pathway web page
Empower your research today using Invitrogen’s comprehensive portfolio of products and services to investigate the Jak/Stat pathway—everything from high-quality reagents for basic research and assay development to validated biochemical and cell-based assays, and world-class profiling and screening services.

Learn more about the Jak/Stat Pathway.

 
Choose the right dead-cell stain for flow cytometry
Accurate interpretation of data obtained by flow cytometry relies on the correct identification and negation of the various nonspecific signals arising from dead cells in the population. Flow cytometrists at Invitrogen recently conducted a survey of 25 membrane stains that have been used to identify dead cells based on membrane integrity. They tested classic membrane-impermeant nucleic acid dyes, monomeric cyanine dyes, annexin V dyes, and amine-reactive dyes. All dyes were tested with a mixture of live and heat-killed cells, before and after formaldehyde fixation, with aged cultures, and with apoptotic cells. Their results demonstrate the utility of optimizing the concentration of dead-cell stain for each experiment and the importance of understanding how a particular dye performs in the intended application.

View the Invitrogen 2008 ISAC poster here.
 
The Total Luminex® 200™ System
Invitrogen has pioneered one of the most comprehensive analyte panels in the industry, and now offers the complete solution by introducing the Luminex® 200™ detection system. Experience the power of Luminex® multiplex analysis all from Invitrogen. The Total Luminex® 200™ System is a compact analyzer that performs up to 100 assays simultaneously in a single well of a microtiter plate. This system is a flexible analyzer based on the principles of flow cytometry, which integrates key xMAP® detection components such as lasers, optics, advanced fluidics, and high-speed digital signal processors. Utilizing the xPONENT® software of the xMAP® technology operating system, the Luminex® 200™ System offers optional technical controls for 21 CFR Part 11 compliance to provide an electronic audit trail.

For more information, visit www.invitrogen.com/luminexinstrument.
 

The Life Science Industry Awards have recognized top companies since 2002, and recipients are selected by an independent survey of life science customers. This year, Invitrogen was a finalist in 12 categories and received the most awards of any company, winning in the following categories:

  • Cell Biology Kits and Reagents
  • Cell Culture Media and Reagents
  • Most Responsive Customer Service
  • Most Knowledgeable Technical Support
  • Most Useful Website
  • Most Memorable Print Advertisements


The awards are hosted by The Scientist magazine and BioInformatics, LLC with the support of The Life Science Executive Exchange and The Science Advisory Board.



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