In This Issue

FEATURED NEW PRODUCTS

Chemiluminescence ELISA Kits Better Sensitivity and Dynamic Range—New Chemiluminescence ELISA Kits
   
LIVE/DEAD® Cell Imaging Kit Imaging Dead and Alive—LIVE/DEAD® Cell Imaging Kit
   
Probes for Synaptic Vesicles New Probes for Synaptic Vesicles—Synaptobrevin, Syntaxin, and Synaptojanin I
   
JAM-A ABfinity A New Antibody for Studying Cell Junctions—JAM-A ABfinity™ Recombinant Rabbit Oligoclonal Antibody
   
Gibco® Microbiological Media Ready-to-Use Microbiological Media—New Gibco® Media for Bacterial and Yeast Cultures
   
New Antibodies See all of this month's New Antibodies

NEW APPLICATIONS

Ion Sphere™ ParticlesQuality Control Assay for Ion Torrent™ Semiconductor Sequencing—Enabling New Workflows With the Qubit® 2.0 Fluorometer

PROVEN PERFORMERS

Streptavidin R-PE Molecular Probes® Streptavidin Phycoerythrin (R-PE)—Consistent Quality for Over 15 Years

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OTHER PUBLICATIONS

BioProbes® Journal of Cell Biology Applications
BioProbes 66 
 
The Molecular Probes® Handbook
Molecular Probes Handbook
 

 

FEATURED NEW PRODUCTS

Better Sensitivity and Dynamic Range—New Chemiluminescence ELISA Kits

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).

 

Chemiluminescence ELISA Kits 
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.



Imaging Dead and Alive—LIVE/DEAD® Cell Imaging Kit

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.

 

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.
Product Quantity Cat. No.
LIVE/DEAD® Cell Imaging Kit, 488/5701 kitR37601



New Probes for Synaptic Vesicles—Synaptobrevin, Syntaxin, and Synaptojanin I

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.

 

Probes for Synaptic Vesicles 
Western blot of rat hippocampal lysate showing specific immunolabeling of the ~145 kDa synaptojanin I protein.



A New Antibody for Studying Cell Junctions—JAM-A ABfinity™ Recombinant Rabbit Oligoclonal Antibody

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.

 

JAM-A ABfinity 
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.



Ready-to-Use Microbiological Media—New Gibco® Media for Bacterial and Yeast Cultures

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.

 

Gibco® Microbiological Media 
Gibco® microbiological media. LB Broth, Terrific Broth, M9 Minimal Salts medium, and YPD Broth.

 

NEW APPLICATIONS

Quality Control Assay for Ion Torrent™ Semiconductor Sequencing—Enabling New Workflows With the Qubit® 2.0 Fluorometer

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.

Ion Sphere™ Particles  
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.

 

PROVEN PERFORMER

Consistent Quality for Over 15 Years—Molecular Probes® Streptavidin Phycoerythrin (R-PE)

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.

 

Streptavidin Phycoerythrin (R-PE) 
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.

 

DEPARTMENTS

On the Web

Gibco® Cell Culture Basics

 

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.


 

Qubit® 2.0

 

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.

Imaging Corner

Imaging Corner
Click to enlarge
 

Fluorescence Labeling of Human Fibroblasts

Neonatal 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.

From the Bench

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.

Methods
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. 

Results
(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.

  

Product Quantity Cat. No.
Click-iT® EdU Alexa Fluor® 488 Imaging Kit, for 50 coverslips1 kitC10337
Click-iT® EdU Alexa Fluor® 555 Imaging Kit, for 50 coverslips1 kitC10338
Click-iT® EdU Alexa Fluor® 594 Imaging Kit, for 50 coverslips1 kitC10339
Click-iT® EdU Alexa Fluor® 647 Imaging Kit, for 50 coverslips1 kitC10340
Click-iT® HPG (L-homopropargylglycine), for nascent protein synthesis5 mgC10186
Click-iT® Protein Reaction Buffer Kit1 kitC10276

 

Molecular Probes® Webinar Series

Molecular Probes Webinar Series

 

Free Webinar—Fluorophore Selection in Experimental Design

Choosing fluorophores is one of the first important decisions you need to make when developing an experiment. This webinar explores the basic characteristics of fluorophores and provides information about online tools that can help you choose the right fluorophores and develop the best assay for your imaging needs.

Date: Thursday, April 26, 2012
Times:
7:00 a.m. and 11:00 a.m. PT

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