In This Issue
FEATURED NEW PRODUCTS
|Better Sensitivity and Dynamic Range—New Chemiluminescence ELISA Kits|
|Imaging Dead and Alive—LIVE/DEAD® Cell Imaging Kit|
|New Probes for Synaptic Vesicles—Synaptobrevin, Syntaxin, and Synaptojanin I|
|A New Antibody for Studying Cell Junctions—JAM-A ABfinity™ Recombinant Rabbit Oligoclonal Antibody|
|Ready-to-Use Microbiological Media—New Gibco® Media for Bacterial and Yeast Cultures|
|See all of this month's New Antibodies|
|Quality Control Assay for Ion Torrent™ Semiconductor Sequencing—Enabling New Workflows With the Qubit® 2.0 Fluorometer|
|Molecular Probes® Streptavidin Phycoerythrin (R-PE)—Consistent Quality for Over 15 Years|
On the Web
From the Bench
|Molecular Probes® Cell Imaging on YouTube
Learn about applications from current customers, see time-lapse videos, watch detailed instrument demos, and hear from our scientists in the lab.
|BioProbes® Journal of Cell Biology Applications|
|The Molecular Probes® Handbook|
FEATURED NEW PRODUCTS
what they are
Two new chemiluminescence ELISA kits for IL-6 and IL-1β offer better sensitivity and a larger dynamic range than conventional colorimetric ELISA kits. These advantages enable detection of proteins at low levels and minimize the need for sample dilutions.
what they offer
- High sensitivity—ability to measure basal levels in serum and plasma samples
- Large dynamic range—broad assay range to minimize guesswork in sample dilutions
- Consistent results—reliable lot-to-lot data
how they work
The total assay incubation time is only 3.5 hours. A monoclonal capture antibody is coated onto the wells of the 96-well plate, and samples are then incubated. After washing away unbound protein, the alkaline phosphatase (AP)-conjugated detector antibody is added, which binds to the antigen at a second site. After washing, a chemiluminescent substrate is added. The relative luminescence units (RLU) can be read on a microplate reader with chemiluminescence capability (luminometer).
- Learn more about Immunoassays
New chemiluminescence ELISA kits outperform colorimetric ELISAs. The standard curves of the Hu IL-1β Chemi ELISA Kit and the ultrasensitive ELISA kit are shifted to the left of the colorimetric ELISA curve, enabling measurement at lower protein concentrations. Also note the larger dynamic range of the chemiluminescence ELISA kit.
|Human IL-6 Chemi ELISA Kit||96 tests||KHC0069|
|Human IL-1β Chemi ELISA Kit||96 tests||KHC0019|
what it is
The new LIVE/DEAD® Cell Imaging Kit is a sensitive, two-color fluorescence cell viability assay optimized for FITC and Texas Red® filters. Quick and easy to use, the kit provides simultaneous determination of live and dead cells with two probes that measure recognized parameters of cytotoxicity and cell viability—intracellular esterase activity and plasma membrane integrity.
what it offers
- Simultaneous detection of live and dead cells
- Convenient, fast, and accurate detection
- Sensitive probes ideal for FITC and Texas Red® filters
how it works
The LIVE/DEAD® Cell Imaging Kit includes a cell-permeant component for staining live cells and a cell-impermeant component for staining dead and dying cells, which are characterized by compromised cell membranes. The LIVE/DEAD® Cell Imaging Kit components are optimized for common imaging filters (FITC and Texas Red®). The live-cell dye produces an intense, uniform green fluorescence in live cells (excitation/emission maxima ~495/515 nm). The red dead-cell dye (excitation/emission maxima ~570/602 nm) predominantly stains nuclei in cells with compromised cell membranes, which are a strong indicator of cell death and cytotoxicity.
Learn more about the LIVE/DEAD® Cell Imaging Kit
Fluorescence staining of live and dead cells. HepG2 cells grown in 96-well microplate wells were stained with the LIVE/DEAD® Cell Imaging Kit and imaged on a Nikon Eclipse® T200 microscope. Live cells are stained green, and dead cells are stained red.
what they are
Two new mouse monoclonal primary antibodies are selective for antigens derived from human brain extracts and specific to synaptic vesicles in a broad range of mammalian species. The SP10 IgM clone is selective for ~16 kDa synaptobrevin, and the SP8 IgG1 clone binds to ~36 kDa syntaxin. The 5H1 IgG1 clone selective for synaptojanin I, a phosphatidylinositol phosphatase, was derived from the C-terminal amino acid sequence from rat.
what they offer
- Validated for immunohistochemistry (IHC), immunocytochemistry (ICC), and western blotting
- Choose from a wide range of IgG1- and IgM-selective secondary antibodies
how they work
Synaptobrevin, also known as VAMP (Vesicle-Associated Membrane Protein), syntaxin, and SNAP-25 form the SNARE complex that mediates vesicle fusion during neurotransmitter release. Using secondary antibodies selective for mouse IgM and IgG1, the anti-synaptobrevin and anti-syntaxin antibodies may be colocalized on tissue samples. Synaptojanin I, which is involved in clathrin-mediated endocytosis of synaptic vesicles, exists in two variant spliced forms: the ~145 kDa isoform is expressed exclusively in neurons, while the ~170 kDa isoform is expressed in non-neuronal peripheral tissues. This clone does not bind to mouse synaptojanin.
- Learn more about Antibodies and Products for Western Blotting
Western blot of rat hippocampal lysate showing specific immunolabeling of the ~145 kDa synaptojanin I protein.
|Synaptobrevin (VAMP), Mouse Monoclonal Antibody||100 µL||P21941|
|Synaptojanin I, Mouse Monoclonal Antibody||100 µL||P21942|
|Syntaxin, Mouse Monoclonal Antibody||100 µL||P21943|
what it is
Junctional adhesion molecule-A (JAM-A) is a tight junction–associated protein that colocalizes with the tight junction proteins occludin, ZO-1, and cingulin. JAM-A has been characterized as a member of the immunoglobulin (Ig) superfamily. This protein is localized at tight junctions in endothelial and epithelial cells, and its subcellular distribution is modulated by inflammatory cytokines. JAM-A is also expressed on circulating leukocytes and platelets, as well as in lymphoid cells.
what it offers
- Consistent, reproducible results
- Minimize the need to revalidate working antibody dilutions each time you order
how it works
ABfinity™ antibodies are manufactured by transfecting mammalian cells with high-level expression vectors containing immunogen-specific heavy- and light-chain antibody cDNA. This production process offers consistent lot-to-lot antibody performance. ABfinity™ oligoclonal antibodies are a mixture of recombinant monoclonal antibodies. This combines the improved signal strength that can come from using a polyclonal, with the highly reproducible results you get from ABfinity™ monoclonal antibodies.
- Learn more about ABfinity™ Recombinant Antibodies
- Find new antibodies every month
- Search for antibodies
Immunocytochemical analysis of U2OS cells stained with JAM-A Rabbit pAb (22HCLC) ABfinity™ Recombinant Oligoclonal Antibody. (A) Alexa Fluor® 488 goat anti–rabbit IgG was used as secondary antibody (green). (B) DAPI was used to stain the nucleus (blue). (C) Composite image of cells showing localization of JAM-A at tight junctions.
|JAM-A Rabbit Recombinant Oligoclonal Antibody – Purified||100 µg||710302|
what they are
Life Technologies now offers Gibco® liquid microbiological media for bacterial and yeast cultures—LB Broth, Terrific Broth, and M9 Minimal Salts medium for bacterial cultures, and YPD Broth for yeast cultures. These traditional bacterial and yeast culture media are available in ready-to-use liquid format for convenience.
what they offer
- Choice—selection of Gibco® media to suit your applications
- Convenience—find media, selection antibiotics, and IPTG all in one place
- Simple—ready-to-use liquid format
how they work
LB Broth is the most referenced bacterial medium for growth and maintenance of recombinant E. coli strains. Terrific Broth is nutritionally rich for higher-density growth and maintenance of recombinant E. coli strains. M9 Minimal Salts medium is commonly used for cultivation and maintenance of E. coli strains. YPD Broth is commonly used for maintenance and propagation of P. pastoris and S. cerevisiae yeast strains.
- Learn more about Microbiological Media
Gibco® microbiological media. LB Broth, Terrific Broth, M9 Minimal Salts medium, and YPD Broth.
|LB Broth (1X), Liquid||1 L||10855-001|
|LB Broth (1X), Liquid||500 mL||10855-021|
|M9 Minimal Salts (2X), Liquid||1 L||A13744-01|
|Terrific Broth, Liquid||1 L||A13743-01|
|YPD Broth, Liquid
Powered by Ion Torrent™ semiconductor chip technology, the Ion Personal Genome Machine™ (PGM™) Sequencer is a benchtop next-generation instrument that offers fast, affordable sequencing. This system sequences DNA templates that have been clonally amplified on primer-coated beads known as Ion Sphere™ Particles (ISPs). Traditionally, confirmation of template-positive ISPs before a sequencing run has been performed using microscopy or flow cytometry. However, systematic quality control of the templated beads has largely been ignored due to the cost involved.
Quality Control of Ion Sphere™ Particles (ISPs)
A new assay using the Qubit® 2.0 Fluorometer enables quick and easy assessment of template-positive ISPs before performing a sequencing run. All ISPs bind a complementary green-fluorescent Alexa Fluor® 488 dye–labeled oligonucleotide, whereas only those beads that are template-positive bind a complementary red-fluorescent Alexa Fluor® 647 dye–labeled oligonucleotide. The Qubit® 2.0 Fluorometer measures the fluorescence emission of both dyes, and an Excel® template is used to calculate the percentage of template-positive ISPs. This quality control step helps determine the likelihood of a successful sequencing run, saving time and expense. The assay is described in the template preparation user guides for the Ion semiconductor sequencing workflow, which can be found on the Ion Community website. Firmware and files for this assay can be downloaded from the Ion Community using the USB drive provided with the Qubit® 2.0 Fluorometer.
- Learn more about the Ion PGM™ Sequencer
The Ion Sphere™ Particle (ISP) assay principle using Alexa Fluor® dyes. The ratio of fluorescence signals from Alexa Fluor® 488 and Alexa Fluor® 647 dye conjugates is used to determine the relative amount of template-positive particles.
|Qubit® 2.0 Fluorometer||1 each||Q32866|
|Ion Personal Genome Machine (PGM™) System||1 instrument||4462921|
|Ion OneTouch™ 200 System Template Kit
|Ion OneTouch™ System||1 system||4470001|
|Ion Sphere™ Quality Control Kit||1 kit||4468656|
Streptavidin phycoerythrin (also known as R-PE streptavidin or SAPE) is typically used for flow cytometry, microarrays, and other applications using biotinylated targets that require either high sensitivity or simultaneous multicolor detection. The sensitivity of R-PE streptavidin is usually 5–10 times greater than that of fluorescein streptavidin conjugates.
Streptavidin phycoerythrin offers:
- Large Stokes shifts with extremely high emission quantum yields, which translate to very bright fluorescent molecules
- Fluorescence not quenched by external reagents, due to protection of the fluorophore by the protein backbone
- Very high water solubility
Molecular Probes® streptavidin R-PE conjugates are purified by HPLC to remove most aggregates and free R-phycoerythrin, minimizing background and improving the signal-to-noise ratio. Our HPLC methods also allow us to achieve high lot-to-lot consistency, which means highly reproducible results from your experiments.
- Learn more about Streptavidin Phycoerythrin
Analytical size-exclusion chromatograms. Chromatograms of free streptavidin, detected by absorption at 280 nm (red curve), and R-phycoerythrin streptavidin, detected by absorption at 565 nm (blue curve), demonstrate that the R-phycoerythrin conjugate is substantially free of unconjugated streptavidin.
|Streptavidin, R-phycoerythrin conjugate (SAPE)||1 mL||S866|
|Streptavidin, R-phycoerythrin conjugate (SAPE), premium grade||1 mL||S21388|
Gibco® Cell Culture Basics
The Gibco® Cell Culture Basics Handbook and Video Library provide an introduction to cell culture, covering topics such as laboratory setup, safety, and aseptic technique. You'll also find information about basic methods for passaging, freezing, and thawing cultured cells.
Watch the Popular Qubit® 2.0 Fluorometer in Action
Over 2,500 fans and counting—the Qubit® 2.0 Fluorometer makes quantitating DNA, RNA, or protein easy, accurate, and fun.
Click to enlarge
Fluorescence Labeling of Human FibroblastsNeonatal human dermal fibroblasts were grown on coverslips, fixed with 4% formaldehyde, and permeabilized with 0.2% Triton X-100. Tubulin was detected with anti–tubulin-α (bovine) antibody (mouse IgG1, monoclonal 236-10501) and visualized using Alexa Fluor® 594 goat anti–mouse IgG. Actin filaments were labeled with Alexa Fluor® 488 phalloidin, and nuclei were stained with Hoechst 33342. Coverslips were mounted on slides using ProLong® Gold antifade reagent. The multiple-exposure image was acquired on a Zeiss LSM 710 confocal microscope at 63x and 16-slice Z-stack maximum intensity projection. Image contributed by Kevin Chambers, Life Technologies.
Elucidating the Regulatory Pathway Components of Asthma
Mohamed JS, Hajira A, Li Z, Paulin D, Boriek AM (2011) Desmin Regulates Airway Smooth Muscle Hypertrophy Through Early Growth-Responsive Protein-1 and MicroRNA-26a. J Biol Chem 286:43394.
The bronchospasms associated with asthma are caused by intrinsically abnormal airway smooth muscle cells (ASMCs). Both hyperresponsiveness and airway remodeling, characteristics of severe asthma, are caused at least in part by hypertrophy (increase in cell size) and hyperplasia (increase in cell number) of ASMCs. Desmin, a cytoskeletal protein and type III intermediate filament expressed in all types of muscle cells, influences essential ASMC functions, including contractile responsiveness.
To investigate the role of desmin in ASMC homeostasis and hypertrophy, Mohamed and colleagues compared ASMCs from desmin-null mice (Des-/-) to primary ASMCs from normal mice (Des+/+). Gene expression profiling and knockdown experiments were used to determine whether desmin induces ASMC hypertrophy through a microRNA-26a (miR-26a) pathway. To measure cell proliferation (DNA synthesis) and nascent protein synthesis, the researchers employed Click-iT® EdU and Click-iT® HPG assays, respectively. Click technology, a bioorthogonal coupling reaction based on an azide–alkyne cycloaddition, provides an alternative to more difficult and time-consuming traditional methods that may require radioactivity.
(Des-/-) ASMCs exhibited hypertrophy and up-regulation of miR-26a. Knockdown of miR-26a in (Des-/-) ASMCs inhibited hypertrophy, while gene delivery and expression of miR-26a in (Des+/+) ASMCs induced hypertrophy. Hypertrophy in (Des-/-) ASMCs was determined by measuring cell size, proliferation, protein synthesis, and protein/DNA ratio compared to ASMCs from wild type (Des+/+) mice. With additional analyses, the authors demonstrated that induction of ASMC hypertrophy was driven by the Erk-1/2/Egr-1/miR-26a/GSK-3β pathway; loss of desmin activates Erk-1/2, leading to up-regulation of miR-26a through the early responsive protein-1 (Egr-1), and the anti-hypertrophic protein glycogen synthase kinase-3β (GSK-3β) is a downstream target of miR26a. The authors concluded that desmin plays an essential role in maintaining a functional ASMC phenotype and is involved in microRNA regulation.
- View the Bibliography Reference
Learn more about Click-iT® Technology
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