- Read the latest issue of the ProbesOnline E-Newsletter.
In This Issue
Single-Cell Analysis — Applied Biosystems® Attune™ Acoustic Focusing Cytometer
||Isolate Newly Synthesized RNA for Expression Analysis — Click-iT® Nascent RNA Capture Kit
Fluorescence-Based Tools for Visualizing Autophagy — LC3B Antibody Kit for Autophagy
Cell-Based Assays Made Easier — BacMam 2.0 GFP Transduction Control
New Antibody Selection Tools from Invitrogen
Quantum Dots for Single-Molecule Tracking
Buzzworthy — High Sensitivity Detection of Cancer In Vivo
- The View — Mouse Intestine Visualized With Alexa Fluor® Dyes
Proven Performers — TC-FlAsH™ for In-Gel Expression Analysis
- On the Web — Antibodies and Apoptosis
Check out the latest issue of BioProbes
FEATURED NEW PRODUCTS
By combining the latest advances in acoustic focusing technology with single-cell analysis, the Applied Biosystems® Attune™ Acoustic Focusing Cytometer gives you optimized performance and throughput without sacrificing sensitivity or accuracy.
what it offers
- Analyze rare cell events faster
- Run more cells in less time, without loss of sensitivity
- Lower variability in population data, better peak separation
The Attune™ Acoustic Focusing Cytometer uses ultrasound waves (>2 MHz) to position cells into a single focused line along the central axis of a sample capillary, resulting in reduced data variability compared to traditional flow cytometers. The Attune™ cytometer also can analyze up to 1,000 µL/min of sample in high-sensitivity mode, which slows the flow rate to ¼ of that of standard cytometers. As a result, cells are exposed to the laser 4 times longer, allowing for higher sensitivity.
Learn More at www.appliedbiosystems.com/
|Applied Biosystems® Attune™ Acoustic Focusing Cytometer.
The Click-iT® Nascent RNA Capture Kit facilitates the capture of newly synthesized (nascent) RNA transcripts from cells or tissue. The captured nascent RNA can be subjected to cDNA synthesis for downstream analysis of gene expression, including real-time PCR, microarrays, and sequencing.
what it offers
- Investigate RNA turnover
- Simple procedure—no antibodies, no radioactivity
- Compatible with downstream high-resolution gene expression applications
- Does not harm cells or RNA
Cells, tissue explants, or animals are fed ethynyl uridine (EU), which is incorporated into nascent RNA. Total RNA or transcriptome RNA can then be extracted using conventional methods. The EU-labeled RNA is then biotinylated using a simple, efficient bioorthogonal click reaction. The biotinylated nascent RNA is captured onto streptavidin magnetic beads and can then be used as template for cDNA synthesis.
Learn More about the Click-iT® Nascent RNA Capture Kit
|The Click-iT® Nascent RNA Capture Kit workflow.|
what it is
Autophagy is the process by which cellular components (including proteins and organelles) are sequestered and degraded by the lysosomal machinery. The LC3B protein plays a critical role in autophagy, and its localization can be used as a general marker for this vital process. The LC3B Antibody Kit for Autophagy enables visualization of autophagy through the detection of LC3B localization.
what it offers
- Robust—enables accurate and sensitive detection of LC3B, with a strong signal compared to other LC3B antibodies
- Flexible—compatible with any anti-rabbit secondary antibody
- Complete—the only LC3B antibody kit that includes an autophagosome inducer
how it works
The LC3B Antibody Kit for Autophagy includes a rabbit polyclonal antibody against LC3B that has been validated for fluorescence microscopy and high-content imaging and analysis. The kit also includes chloroquine—following chloroquine treatment, lysosomal pH increases and the normal autophagic flux is disrupted, resulting in autophagosome accumulation.
- Learn More about Autophagy
|LC3B Antibody Kit for Autophagy.
Read More +
HeLa, A549, and HepG2 cells were treated with 50 µM chloroquine, which blocks fusion of autophagosomes with lysosomes, causing an accumulation of autophagosomes. Following fixation and permeabilization, cells were stained with anti-LC3B rabbit polyclonal antibody and visualized with Alexa Fluor® 647 goat anti–rabbit IgG. The intensity of labeling was quantified on autophagosomes or the adjacent cytosol using Slidebook™ digital microscopy software (A), but can also be imaged directly (B, HeLa cells).
The BacMam system uses a modified baculovirus as a vehicle to efficiently deliver and express genes in mammalian cells. The popular BacMam platform has been improved to enable more efficient cell entry, and includes stronger promoter elements that boost expression levels. Cell types poorly transduced with earlier BacMam versions are now transduced quantitatively with a simple, one-step process. Try it in your system with the BacMam GFP Transduction Control.
what it offers
- Efficient—>90% transduction of a wide range of mammalian cell lines
- Fast and convenient—simply add BacMam 2.0 to cells, incubate overnight, and assay
- Safe—nonreplicating in mammalian cells, lack of observable cytopathic effects, and biosafety level 1 handling
Just mix the BacMam 2.0 GFP Transduction Control with your cells at the time of plating on a coverslip or microwell plate, incubate, and measure transduction efficiency the next day. A range of BacMam-based reagents are available, including Cellular Lights™ imaging reagents, Premo™ autophagy and cell cycle sensors, and pathway analysis kits. You can easily try the power of BacMam 2.0 with our convenient trial-size vials.
Learn More about the BacMam System
|More efficient transduction with BacMam 2.0 in primary and stem cells.
Read More +
Comparison of the transduction efficiency of BacMam 1.0 GFP (top row) and BacMam 2.0 GFP (bottom row) in T84 adenocarcinoma cells (A) and adipose-derived stem cells (ADSC) (B), under identical conditions.
Qdot® nanocrystals are ideal for live-cell studies of extracellular components such as receptors, and their photostability and brightness permit prolonged detection of single molecules. In a recent example, Geng et al.  show that streptavidin Qdot® 655 nanocrystals bound to biotinylated α-bungarotoxin can be used to visualize and track individual acetylcholine receptors on living cells as they assemble into aggregates. They also show by way of comparison that standard dyes (like Alexa Fluor® 488 dye) can only resolve the large aggregates of preformed clusters, leaving the individual receptor subunits undetected (Figure 1). The staining was as easy as adding, washing, and imaging.
High sensitivity despite the use of suboptimal filters
It should be noted that this high sensitivity was achieved despite using suboptimal filters for Qdot® nanocrystal visualization. A rhodamine filter set, with 510–550 nm excitation and a longpass filter at >590 nm, was used. With an excitor closer to the excitation maximum of 300 nm (such as a typical DAPI excitor (350 nm/50 nm)), an 8–10 times brighter signal could have been seen. However, the signal the authors attained was more than bright enough and should encourage researchers anxious to try Qdot® nanocrystals in their experiments to use standard-fluorophore filter sets.
Streptavidin and biotin—the foundation of labeling and detection
Streptavidin and biotin are the foundation of many bioconjugates for labeling and detection. Simple, fast, and nearly covalent (K ≈ 10–21 ), the reagents are also very adaptable. For example, any color of streptavidin Qdot® nanocrystal can be used with any biotinylated biomolecule. The same is true for streptavidin Alexa Fluor® dyes. We offer a wide range of biotinylated biomolecules, including cholera toxin, epidermal growth factor, α-bungarotoxin, phalloidin, and transferrin.
|1.||Geng L, Zhang HL, Peng HB (2009) The formation of acetylcholine receptor clusters visualized with quantum dots. BMC Neurosci 10:80.|
|2.||Launer HF, Fraenkel-Conrath H (1951) The avidin-biotin equilibrium. J Biol Chem 193(1):125–132.|
|α-Bungarotoxin, biotin-XX conjugate||500 µg||B1196|
|Cholera Toxin Subunit B (recombinant), biotin-XX conjugate||100 µg||C34779|
|Epidermal Growth Factor, biotin-XX conjugate (biotin EGF)||20 µg||E3477|
|Biotin-XX Phalloidin||50 units||B7474|
|Transferrin from human serum, biotin-XX conjugate||5 mg||T23363|
|Endogenous Biotin-Blocking Kit, 100 assays||1 kit||E21390|
|Qdot® 655 Streptavidin Conjugate||200 µL||Q10121MP|
|Qdot® 655 Streptavidin Conjugate, starter kit||50 µL||Q10123MP|
|Streptavidin, Alexa Fluor® 488 conjugate||1 mg||S11223|
High sensitivity detection of cancer in vivo using a dual-controlled activation fluorescent imaging probe based on H-dimer formation and pH activation
Mikako Ogawa, Nobuyuki Kosaka, Celeste A. S. Regino, Makoto Mitsunaga, Peter L. Choyke, and Hisataka Kobayashi. Mol BioSystems 2010
Driven by the desire to improve the target-to-background ratio (TBR) of molecular imaging reagents, Ogawa et al. investigated the xanthene fluorophore TAMRA™, SE as a cancer-detecting reagent. Introduction of avidin-TAMRA™ conjugates enabled visualization of D-galactose receptor–positive tumors with high TBR 2 hours after the probe was injected. A small amount of fluorescence remained, however, even in the quenched state. To improve this, the team repeated the experiments using the pH-dependent fluorescent pHrodo™ dye, which forms quenched H-type dimers similar to those formed by TAMRA™. When injected into tumor-bearing mice, the avidin–pHrodo™ dye conjugate enabled visualization of the tumors with high TBR 1 hour and 2 hours after injection. The improved TBR was attributed to the dual control of the probe: pH activation and H-dimer quenching. In contrast, the avidin-TAMRA™ conjugate also depicted tumors 1 hour and 2 hours after injection, but the TBR was lower due to higher nonspecific binding.
View the bibliography reference
- Learn More about pHrodo™ Indicators
|TAMRA™, SE||25 mg||C1171|
|pHrodo™, SE||1 mg||P36600|
Mouse intestine cryosection showing basement membranes labeled with chicken IgY anti-fibronectin primary antibody and Alexa Fluor® 647 goat anti–chicken IgG secondary antibody (pseudocolored purple). Goblet cells and crypt cells were labeled with Alexa Fluor® 594 Wheat Germ Agglutinin (red). The microvillar brush border and smooth muscle layers were visualized with Alexa Fluor® 488 Phalloidin (green). Nuclei were counterstained with SYTOX® Blue Nucleic Acid Stain (blue).
|Alexa Fluor® 647 Goat Anti–Chicken IgG Secondary Antibody||0.5 mL|
|Alexa Fluor® 594 Wheat Germ Agglutinin||5 mg|
|Alexa Fluor® 488 Phalloidin||300 units|
|SYTOX® Blue Nucleic Acid Stain||250 μL|
TC-FlAsH™ for In-Gel Expression Analysis
The TC-FlAsH™ in-gel expression analysis kits allow rapid and easy confirmation of the presence of proteins of interest that are tagged with a tetracysteine (TC) tag. The green fluorescence of the TC-FlAsH™ reagent is paired with a contrasting stain in either red or orange that allows visualization of your total protein alongside the BenchMark™ total protein ladder for reference.
What it offers:
|Rapid, direct labeling of purified or crude cell lysate—no gel staining needed|
|In-gel visualization—or even within the cassette—of tagged and total protein|
|Westerns are no longer necessary to confirm TC-tagged proteins|
How it works:
FlAsH is a cell-permeable fluorescein derivative that emits bright green fluorescence when bound to tetracysteine (TC) sequences. The most commonly used TC is the six amino acid Cys-Cys-Pro-Gly-Cys-Cys sequence. As this sequence rarely appears in endogenous proteins, incorporating the sequence into target proteins generates a small but highly specific target for protein labeling.
- Learn More about TC FlAsH™ Expression Analysis Detection Kits
FlAsH detection of TC-tagged proteins expressed in E. coli. (A) TC-tagged protein labeling with FlAsH in-gel detection; before, 30 min, and 4 hr post-induction of protein expression (RNAPα N-term CCPGCC (lanes 2, 3, and 4), RNAPα WT (lanes 5, 6, and 7), CaM internal CCEQCC (lanes 8, 9, and 10), and GFP C-term CCRECC (lanes 11, 12, and 13) and resolved on a Tris-glycine 4–20% gradient gel. (B) Total protein detection.
|TC-FlAsH™ Expression Analysis Detection Kit – Orange||1 kit||A10067|
|TC-FlAsH™ Expression Analysis Detection Kit – Red||1 kit||A10068|
|Finding the Right Primary and Secondary Antibodies
Finding primary and secondary antibodies has never been easier. With our improved search tools for primary and secondary antibodies, you can quickly and easily select the optimal antibody for your research. A simple search by target yields specific results that are quickly narrowed down by filters that you specify, such as application or species reactivity.
Explore antibodies for your research today!
|New Apoptosis Application Web Page
Whether you're measuring annexin translocation or caspase activation, the new Apoptosis page will help you find the products and information you need to better understand programmed cell death. Check out our reagents and kits for multiparametric detection of apoptosis today!
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