ProbesOnline October, 2011

Read the latest issue of the ProbesOnline E-Newsletter.

In This Issue


New Mouse Immunoglobulin Isotyping Kit   Identify Mouse Antibody Subtypes Reliably — New Mouse Immunoglobulin Isotyping Kit
pHrodo™ Red Labeling Kit and pHrodo™ Red Avidin   Fluorogenic pH Sensors for Live Cell AnalysespHrodo™ Red Labeling Kit and pHrodo™ Red Avidin
LRRK2 ABfinity™ Antibodies   New Recombinant Antibodies — Leucine-Rich Repeat Kinase 2 (LRRK2) ABfinity™ Antibodies
phospho-p70S6 Kinase Recombinant Oligoclonal Antibody   Investigate p70S6 Kinase Up-regulation — New Phospho-p70S6 Kinase [pT421/pS424] Recombinant Oligoclonal Antibody




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Identify Mouse Antibody Subtypes Reliably—New Mouse Immunoglobulin Isotyping Kit

what it is
The Invitrogen™ Mouse Immunoglobulin Isotyping ELISA Kit enables rapid identification of mouse immunoglobulin isotypes IgG (G1, G2a, G2b, G3), IgA, and IgM from purified antibodies, secreted antibodies from hybridomas, transfected cell culture supernatants, and ascites fluid. The kit is designed for experimental flexibility and contains reagents for 96 tests of each immunoglobulin isotype.

what it offers

  • Reliability—rigorous quality validation helps ensure specificity of isotype determination
  • Flexibility—antibodies are provided in separate solutions so that you can design your plate plan as needed
  • Simplicity—all necessary reagents, including controls, are supplied

how it works
The Mouse Immunoglobulin Isotyping Kit uses a solid-phase ELISA format. The mouse immunoglobulins (Ig) are classified based on their heavy (IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, IgD, and IgE) and light (kappa (κ) and lambda (λ)) chains. The kit contains six separate monoclonal capture antibody solutions recognizing the specific isotypes (IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA). You can coat wells of your assay plate with any of the solutions to meet your specific experimental needs. After coating wells with the supplied capture antibodies and blocking the plate, simultaneously add your experimental samples or controls, and biotinylated detector antibodies (either κ or λ), to the wells. Wash the plate, add streptavidin-HRP, wash again, and add the chromogenic substrate. Finally, add the stop solution and read the plates at 450 nm to obtain your results.

Fluorogenic pH Sensors for Live Cells

Suggested 96-well plate plan.
Colored wells indicate expected positive signals using the control mix. Note: Each vial of control in the kit contains IgG1(κ), IgG2a(λ), IgG2b(κ), IgG3(λ), IgA(κ), and IgM(κ) antibodies.
Product Quantity Cat. No.  
Mouse Immunoglobulin Isotyping Kit 6 plates 992000 Order Now

Fluorogenic pH Sensors for Live-Cell Analyses—pHrodo™ Red Labeling Kit and pHrodo™ Red Avidin

what they are
The popular pHrodo™ dye, a fluorogenic pH sensor for endocytosis in live-cell studies, is now offered in two additional formats: as a microscale antibody/protein labeling kit, and as an avidin conjugate for use with any biotinylated target. pHrodo™ dye exhibits pH-sensitive red fluorescence that increases in intensity with increasing acidity. pHrodo™ dye is essentially dark in the extracellular environment but exhibits  bright red fluorescence at pH 5–7 as it undergoes endocytosis. Although we call the dye pHrodo™ Red, these products contain the same pHrodo™ dye as our pHrodo™, SE; pHrodo™ dextran; and pHrodo™ BioParticles® for phagocytosis.

what they offer

  • Flexibility to create your own probes for phagocytosis and endocytosis investigations
  • Compatibility to multiplex with green dyes such as GFP, fluo-4, and calcein
  • Designed and tested for imaging, flow cytometry, and HTS/HCS (microplate) applications

how they work
pHrodo™ Red Avidin and the pHrodo™ Red Microscale Labeling Kit offer a rapid means to make any material, including proteins, peptides, viruses and small molecules a pHrodo™ Red conjugate. The labeling kit includes reagents for 3 labeling reactions of 20–100 µg protein with excellent recovery, and can also be used for labeling viruses. Alternatively pHrodo™ Red Avidin can be used to create bioconjugates using the easy, high-affinity avidin–biotin interaction. Use pHrodo™ Red Avidin when a biotinylated target already exists.

pHrodo™ dye is ideal for detecting changes in pH

pHrodo™ dye is ideal for detecting changes in pH. (
A) pHrodo™ dye exhibits pH-sensitive fluorescence. (B) Endocytic vesicles undergo acidification during the maturation process.

New Recombinant Antibodies—LRRK2 ABfinity™ Antibodies

what they are
Leucine-rich repeat kinase 2 (LRRK2) is phosphorylated at serine 935 in brain tissue and is implicated in familial Parkinson’s disease. When LRRK2 is phosphorylated at serine 935, the 14-3-3 protein binds specifically, and this may be an important mechanism in regulating dopamine physiology.

what they offer
ABfinity™ recombinant monoclonal and oligoclonal antibodies offer consistent results, minimizing the need to revalidate working antibody dilutions for your experiments each time you order.

how they work
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. 
Our LRRK2 phosphoserine 935 ABfinity™ Recombinant Rabbit Antibodies are validated for western blotting and ELISA applications.

LRRK2 ABfinity™ Antibodies

Leucine-Rich Repeat Kinase 2 (LRRK2) ABfinity™ Antibody performance.

Indirect ELISA was performed using the indicated concentrations of LRRK2 phosphoserine 935 [pS935] Rabbit Recombinant Oligoclonal Antibody (Cat. No. 710097) to detect  phospho-LRRK2 peptide coated onto the plate. A nonlinear regression analysis was performed (4 PL), and LOD and LOQ for the antibody were determined to be 450 pg/mL and 1,370 pg/mL, respectively.

Investigate p70S6 Kinase Up-regulation—New Phospho–p70S6 Kinase [pT421/pS424] Recombinant Oligoclonal Antibody

what they are
The ribosomal p70S6 kinase is known for its role in modulating cell-cycle progression, cell size, and cell survival. Activated p70S6 kinase is phosphorylated at threonine 421 and serine 424, and higher phosphorylation levels are associated with neurofibrillary pathology in Alzheimer's disease.

what they offer
ABfinity™ recombinant monoclonal and oligoclonal antibodies offer consistent results, minimizing the need to revalidate working antibody dilutions for your experiments 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.
Phosphorylated p70S6 Kinase [pT421/pS424] Recombinant Rabbit Oligoclonal is validated for western blotting, ELISA, and immunocytochemistry.

phospho-p70S6 Kinase Recombinant Antibody

Immunocytochemistry analysis using the new Phospho-p70S6 Kinase Recombinant Antibody.

HeLa cells were incubated with p70S6 Kinase [pT421/pS424] Rabbit Recombinant Oligoclonal Antibody and detected using Alexa Fluor® 488 goat anti-rabbit secondary antibody (A11008) (green). DAPI was used to stain the nucleus, and actin was visualized using Alexa Fluor® 594 phalloidin (red). (A, B) Composite image of cells showing cytoplasmic and nuclear localization of phosphorylated p70S6K. (C) Competition with the phospho-p70S6K [pT421/pS424] peptide.


CellLight® Reagents in Translational Medicine

CellLight® reagents are ready-to-use BacMam viral particles containing fluorescent protein constructs targeted to specific subcellular structures. They are simple and effective tools for introducing targeted intracellular fluorescent labels into living cells. Simply add the CellLight® reagent to your cells, incubate overnight, and you’re ready to image your cells.

  • Efficient transduction of mammalian cells, including “difficult” cells such as primary and stem cells
  • Robust expression and sustained cellular function of fluorescent proteins for highly specific labeling
  • Easily study dynamic changes, colocalization, and signaling events in living cells
  • Compatible with fixed-cell staining and high-content analysis

In a recent study, Gasparian et al. published results that help elucidate the mode of action of a class of small molecules, curaxins, that have the potential to be useful in cancer therapy (Sci Transl Med 3, 95ra74 (2011)). They found that this newly identified class of anti-cancer compounds exhibits activity in mice at levels that are not toxic or genotoxic—an unusual property for DNA-targeted chemotherapeutics. In addition, they show that these compounds “simultaneously activate p53 and inhibit NF-κB”, proteins that are dysregulated in many malignant tumor types. CellLight® Histone 2B-RFP, BacMam 2.0 was used in this study to help show that curaxins cause functional inactivation of the FACT (facilitates chromatin transcription) complex by causing one of its two subunits, structure-specific recognition protein 1 (SSRP1), to become tightly associated with chromatin.


Easy, Reliable Cell Counting of 120 Different Cell Types and Counting—Countess® Automated Cell Counter

  • Countess® Automated Cell CounterAccurate—without the subjectivity of manual cell counting and user-to-user variability
  • Fast—total cell counts, percent viable cells, and average cell size in as little as 30 seconds
  • Versatile—can be used with a wide variety of cells: documented with 120 different cell types from 18 species so far
  • Convenient—requires no cleaning or routine maintenance, and minimal setup

Have confidence in your cell counts with the Countess® Automated Cell Counter, which has been referenced in over 100 peer-reviewed publications representing over 120 different cell types from 18 species. The Countess® Automated Cell Counter is a benchtop instrument that offers easy and accurate cell and viability counts in as little as 30 seconds. This image-based cell counting instrument eliminates the tedium and subjectivity of manual cell counting and minimizes subjective judgments that can lead to errors. By offering accurate cell count, cell size, and cell viability data, the Countess® instrument aids in improving downstream results, which translates to fewer experimental repeats needed due to subjective errors. To supplement the peer-reviewed publications, we have generated several technical and application notes covering topics such as counting blood, primary cells, and stem cells.

The Countess® Automated Cell Counter continues to set the standard for benchtop cell counters.


On the Web

FLoid™ Cell Imaging Station
Introducing the FLoid™ Cell Imaging Station —
from vessel to image in as few as 2 clicks

Find out how simple it is to collect fluorescent cell images using the new FLoid™ Cell Imaging Station.

Imaging Corner

Live HeLa Cell Imaging  

click to enlarge

Live HeLa cell imaging. Live HeLa cells were transduced with CellLight® Plasma Membrane-GFP, BacMam 2.0 construct (green) and wheat germ agglutinin, Alexa Fluor® 594 conjugate (red), both targeting the plasma membrane. The cells were then counterstained with Hoechst 33342 dye (blue) to label nuclei, and imaged live.    

From the Bench

There have have been several outbreaks of deadly Escherichia coli in the last few years; the US Centers for Disease Control and Prevention (CDC) lists six such events in 2010 and 2011, with the sources of contamination covering a wide range of food products that include beef, lettuce, cheese, and nuts. Shiga toxins produced by certain strains of E. coli and Shigella dysenteriae are responsible for the severe illness and death that can result when humans become infected with these bacteria. It is hoped that research aimed at better understanding the effect of shiga toxins on host cells will help identify strategies for effective treatment and/or prevention of illness. To that end, scientists at the Texas A&M Health Science Center and colleagues recently compared intracellular trafficking and autophagosome formation of shiga toxin in toxin-sensitive and toxin-resistant cells. They were particularly interested in understanding whether autophagy in response to shiga toxin is a protective mechanism or whether it contributes to apoptosis. They found that shiga toxin was translocated to different cell compartments in toxin-sensitive and -resistant cells. In addition, they report that both cell types exhibited autophagosome formation. They determined that “proteolytic cleavage of Atg5 and Beclin-1 plays pivotal roles in switching non-cytotoxic autophagy to cell death signalling.”

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