• protein detection chemistry │ Click-iT® technology │ Click-iT® toolbox reagents
• cell biology│second-messenger detection│Fluo-4 Direct™ Calcium Assay Kit
• flow cytometry │cell cycle analysis │ FxCycle™ dyes
• flow cytometry │control reagents │ ArC™ Bead Kit
• endocytic pathways | targeted fluorescent proteins | Organelle Lights™ and pHrodo™ reagents
• antibodies | primary antibodies | cell signaling antibodies
• cell biology │ fluorescence imaging │plant imaging
• cellular imaging │high-content screening │ cell health and cytotoxicity assays
• proven performers
• buzzworthy
• the view
• on the web
• in the field

Click-iT® toolbox reagents for rapid detection and identification of posttranslational modifications
 
what they are 
With the latest additions to the Click-iT® toolbox of azide- and alkyne-containing biomolecules, you can rapidly discover key, but often elusive, protein posttranslational modifications (PTMs) including:

• fucosylation—an important PTM for glycans that participate in cell–cell interactions and cell migration processes
• prenylation (farnesyl or geranylgeranyl)—important PTMs for protein trafficking
• fatty acylation (myristic acid or palmitic acid)—important PTMs for cell signaling, protein localization, trafficking, stability, and protein–protein interactions

how they work
Click chemistry is a powerful method for joining biomolecules quickly and specifically, and offers significant advantages over standard metabolic labeling and detection methods. An extremely fast reaction, click chemistry involves a copper-catalyzed covalent reaction between an alkyne and an azide, functional groups that do not occur naturally in biological systems. This uniqueness virtually eliminates the nonspecific binding or high background that can compromise other widely used detection methods. The small size of alkynes and azides compared to antibodies, or even biotin, allows for effective modification of small molecules and efficient penetration of cells and tissue by the Click-iT® reagents. Performing a click reaction requires the corresponding dye- or hapten-azide or alkyne and either the Click-iT® Cell Reaction Buffer Kit for analysis by flow cytometry or imaging, or the Click-iT® Protein Reaction Buffer Kit for analysis by standard biochemistry techniques such as gel electrophoresis, western blotting, or mass spectrometry. 

what they offer

• pioneering biological insights—expose previously undiscovered and elusive posttranslational modifications
• rapid results—discover and identify modified proteins in hours or days as opposed to weeks or months with radiolabeling techniques
• context-rich data—easy multiplexing with other detection methods, including immunolabeling, flow cytometry, and even western blotting

Overview of the click chemistry azide/alkyne reaction. The azide and alkyne moieties are interchangeable, where the molecule can be labeled with an alkyne and react with a fluorophore- or hapten-azide.

• compatible with serum-containing media—no removal or wash steps needed
• simple add-and-read format—just load cells, stimulate, and read
• large assay window—low background with high fluorescence intensity
  
  

Dose-dependent calcium response to muscarnic 1 (M1) receptor agonists.

Dose-dependent inhibition of the calcium response by M1 receptor antagonists.

• cell cycle analysis using 405 nm or 633/635 nm excitation sources for more flexibility in experimental design
• minimal compensation with 488 nm excitable dyes for simpler analysis
• tight CVs for accurate results
• packaged as solid dye for longer shelf life and value

• fast and simple bead-based compensation for amine-reactive dyes
• accurate and consistent results
• easy combination with the AbC™ Anti-Mouse Bead Kit for multicolor immunophenotyping
• does not require treating cells for use in compensation setting

Combine Organelle Lights™ and pHrodo™ reagents to visualize endocytosis and protein trafficking in real-time

what it is
A powerful combination of fluorescent protein–based Organelle Lights™ reagents and pH-sensitive pHrodo™ reagent–labeled proteins facilitates visualization of protein internalization and trafficking in live cells.   

how it works
The pHrodo™ dye is a novel, red-emission, pH-sensitive dye that dramatically increases fluorescence as the pH of its surroundings becomes more acidic, making it an ideal tool to study endocytosis and its regulation by drugs and/or environmental factors. Organelle Lights™ reagents are ready-to-use fusion constructs prepackaged in baculovirus particles for highly efficient cellular delivery. Conjugates of the protein of interest (e.g., a receptor) are prepared with the amine-reactive pHrodo™ succinimidyl ester. The lysosomes or endosomes are labeled with the appropriate GFP-based Organelle Lights™ reagent, and overnight incubation allows expression of the targeted fluorescent protein. GFP is the recommended fluorescent protein, as it can be easily distinguished from the red fluorescence of the pHrodo™ dye. Once the cells are ready, add the pHrodo™ conjugate and incubate while imaging the conjugate as it travels through this dynamic cellular pathway.

what it offers

• real-time analysis—pHrodo™ and Organelle Lights™ reagents are live-cell compatible
• content-rich results—pHrodo™ and Organelle Lights™ reagents are amenable to formaldehyde-based fixation and can be multiplexed with blue- and far-red–fluorescent dyes or labeled antibodies

HeLa cells transduced with Organelle Lights™ Endosome-GFP. The following day, cells were washed and then incubated with 10 mg/mL pHrodo™ dextran and 1mg/mL Hoechst 33342 for 5 min. It is possible to visualize endocytosis of pHrodo™ by colocalization of Organelle Lights™ Endosome-GFP and pHrodo™ dextran. Cells were imaged on a Delta Vision Core microscope using standard DAPI/FITC/TRITC/Cy®5 filters.

Learn more about Organelle Lights™ reagents.
Learn more about pHrodo™ reagents.

New validated primary antibodies for oncology, neurobiology, and immunology research

what they are
This month we are launching a diverse array of new cell signaling antibodies covering a broad spectrum of research areas, including oncology, neurobiology, immunology, and DNA modification. These antibodies are highly specific and validated with multiple applications using various species. The high quality of these antibodies is assured by ISO certified manufacturing techniques, and they are guaranteed to work when used as described. 

how they work
We are actively expanding our collection of primary antibodies to enable analysis of key cell signaling targets. These new antibodies include extracellular antibodies against the cytokines IL-23 and IL-17, neuroscience antibodies against Dopamine 1A, anti-acelated lysine antibody to detect histone/transcription factor modification, and a phosphospecific antibody against the tumor suppressor, retinoblastoma protein (Rb).

what they offer

• confidence— all phosphorylation site specificities have been verified with peptide competition
• performance—robust antibodies validated for multiple applications
• selection—large menu of unique specificities, with wide range of targets and modification sites

RB [pS612], rabbit polyclonal antibody: western blot and peptide competition. Extracts of Jurkat cells in high growth phase were resolved by SDS-PAGE and transferred to PVDF. The membrane was blocked with 5% BSA-TBST at room temperature for 1 hr, then incubated with RB [pS612] for 2 hr at room temperature in 3% BSA-TBST, following prior incubation with: no peptide (lane 1), a generic phosphoserine-containing (PS) peptide (lane 2), the non-phosphopeptide (NP) corresponding to the phosphopeptide immunogen (lane 3), or the phosphopeptide (P) immunogen (lane 4). After washing, the membrane was incubated with goat F(ab')2 anti–rabbit IgG alkaline phosphatase (Cat. no. ALI4405) and signals were detected using the Tropix® Western-Star™ detection method. The data show that only the phosphopeptide corresponding to Rb [pS612] blocks the antibody signal, demonstrating the specificity of the antibody.

See a list of additional phosphorylation site–specific antibodies.
To browse antibodies by specificity or application, visit www.invitrogen.com/antibodies.

Fluorescence technologies for cell health and toxicity in high-content imaging and analysis

what they are
We offer a comprehensive set of solutions for determining cell health and cytotoxicity in cell-based assays on automated imaging and high-content screening (HCS) platforms.

how they work
HCS cell health and cytotoxicity assays use a wide variety of fluorescent probes to enable the fixed end-point detection of apoptotic, genotoxic, mitotoxic, and cytotoxic effects of drugs. The assays are compatible with detergent permeabilization and formaldehyde-based fixation, enabling antibody detection of on-target effects in the same cells. With these easy-to-use tools, simultaneous interrogation of multiple aspects of cytotoxicity and cell health is possible, allowing generation of high-content results needed to make the next critical decision in the process of drug discovery and development.

what they offer
The HCS cell health and cytotoxicity assays enable critical insights into various aspects of cell health:

• apoptosis
• cell proliferation
• cell viability
• DNA damage
• mitochondrial health
• mitotic index
• nascent protein synthesis
• phospholipidosis and steatosis

Detection of protein synthesis inhibition using the Click-iT® AHA Alexa Fluor® 488 Protein Synthesis HCS Assay. (A) Dose response curves, performed in duplicate. U2OS cells were treated in L-methionine–free medium containing 50 μM Click-iT® AHA and cycloheximide (85 pM–5 μM) for 30 min. Images show untreated cells (B) and cells treated with 150 μM cycloheximide (C).

Learn more about HCS cell health and cytotoxicity assays at www.lifetech.com/hcs.

Experience the best—Alexa Fluor® dyes

From blue to red and into the near infrared, the Alexa Fluor® series of dyes provides many options for multicolor detection. Conjugates made with Alexa Fluor® dyes exhibit more intense fluorescence than other spectrally similar conjugates and are more photostable than most other fluorescent conjugates, allowing more time for image capture.

The Alexa Fluor® dyes remain the best and brightest conjugates we have ever tested. Recently, several new contenders have become available, most making claims of superior performance both in brightness and photostability. To investigate these claims, we compared all green-fluorescent secondary antibody conjugates available at the time of testing.

For more information about the Alexa Fluor® dye series, visit www.invitrogen.com/alexa.

Multicolor fluorescence image of mouse testis cross section. Spermatozoa were detected using the green-fluorescent Alexa Fluor® 488 conjugate of lectin PNA (Cat. no. L21409). Actin filaments were visualized using red-fluorescent Alexa Fluor® 568 phalloidin (Cat. no. A12380). Nuclei were stained with blue-fluorescent DAPI (Cat. no. D1306, D3571, D21490).