ProbesOnline™ April 2014—New products for live-cell H₂O₂ detection, cell proliferation, plasma membrane staining, and protein labeling

Dynamic measurement of hydrogen peroxide in live cells

Premo™ Cellular Hydrogen Peroxide Sensor

What it is
The new Premo™ Cellular Hydrogen Peroxide Sensor is a fluorescent protein–based reporter that detects hydrogen peroxide (H₂O₂) in living cells in a dynamic and reversible manner. The Premo™ H₂O₂ sensor combines the selectivity of roGFP-Orp1 chimeras for H₂O₂ with the transduction efficiency of BacMam 2.0 technology for ease of use with different cell lines. The sensor can be readily combined with other probes for powerful multiplex assays in cell biology.

What it offers

• High selectivity for hydrogen peroxide
• BacMam 2.0 gene delivery technology allows easy and efficient expression in most cell types
• Dynamic and reversible measurement of hydrogen peroxide in live cells
• Improved accuracy using ratiometric detection

How it works
roGFP is a modified GFP with two cysteines introduced into its β-barrel structure, rendering this protein sensitive to oxidation and useful in probing reactive oxygen species (ROS). Upon oxidation, these cysteines form a disulfide bridge leading to protonation of the roGFP and a shift in the excitation maximum from 488 nm (of normal GFP) to 400 nm. This shift in the excitation maximum permits ratiometric detection of the fluorescence emission at 515 nm. Orp1 is an H₂O₂-sensitive yeast protein that forms disulfides upon highly selective reaction with H₂O₂, and this protein has the ability to transfer signals by disulfide exchange to other redox-sensitive proteins [1] such as  roGFP.  Orp1, expressed as a fusion with roGFP, serves as a “molecular antenna” to convey H₂O₂ selectivity to roGFP in live cells.

1. Gutscher et al. (2009) J Biol Chem 284:31532–31540.

• Download the Premo™ Cellular Hydrogen Peroxide Sensor user manual
  

Time and dose response curves for Premo™ Cellular Hydrogen Peroxide Sensor. U2OS cells were transduced with Premo™ Cellular Hydrogen Peroxide Sensor; after 48 hours, time-lapse imaging was done on a Zeiss® LSM 710 confocal microscope. Images were captured every 15 seconds, over a 10-minute time span after addition of varying doses of H2O2, using alternating 400 nm and 488 nm excitation. Fluorescence emission was captured at 515 nm. Fluorescence intensity values were quantified by marking regions of interest on the cells and were used to calculate 400 nm/488 nm excitation ratios. The ratios were plotted against time.

Improved multiplex compatibility

New Click-iT® Plus EdU proliferation assay kits for flow cytometry

What they are
The Click-iT® Plus EdU flow cytometry kits provide a simplified and more robust assay for analyzing DNA replication in proliferating cells compared to traditional BrdU methods. Unlike the original Click-iT® EdU assay and some BrdU assays, the Click-iT® Plus EdU assay can be used in conjunction with R-PE and R-PE tandems, as well as fluorescent proteins such as GFP and mCherry.

What they offer

• Multiplexable—unlike BrdU or the original Click-iT® EdU, Click-iT® Plus EdU is completely compatible with R-PE, R-PE tandems, and fluorescent proteins
• Accurate—superior results compared to BrdU assays
• Fast—results in as little as 60 minutes

How they work
The Click-iT® Plus EdU proliferation assay is a novel alternative to the BrdU assay. EdU (5-ethynyl-2′-deoxyuridine) is a thymidine analog that is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry, which is a copper-catalyzed covalent reaction between an azide and an alkyne. The Click-iT® Plus reaction uses a modified azide in place of the azide used in the original Click-iT® reaction. As a result of the modification, the concentration of free copper in the sample is significantly lower and fluorescence signals from fluorescent proteins (e.g., R-PE, R-PE tandem dyes, and GFP) are not quenched. The speed and accuracy of the Click-iT® Plus EdU reaction is comparable to that of the original Click-iT® EdU reaction.

• Learn more about Click-iT® Plus EdU assay kits for flow cytometry

Compatibility of Click-iT® Plus EdU reaction with R-PE tandem conjugate fluorescence. Jurkat (human T-cell leukemia) cells were treated with EdU for 2 hours and then stained with a PE-Cy®7 conjugate of mouse anti–human CD3 antibody. Newly synthesized DNA was detected according to the protocol included in the Click-iT® Plus EdU Alexa Fluor® 488 Flow Cytometry Assay Kit. The cells were analyzed on the Attune® Acoustic Focusing Cytometer using 488 nm excitation. The Alexa Fluor® 488 fluorescence signal was detected using a 530/30 nm bandpass emission filter, while the PE-Cy®7 fluorescence signal was detected using a >640 nm longpass filter. The data demonstrate that the PE-Cy®7 fluorescence signal was retained following detection of the incorporated thymidine analog, EdU, with the Click-iT® Plus Alexa Fluor® 488 picolyl azide.

Customizable biomolecule detection

Click-iT® Plus Alexa Fluor® Picolyl Azide Toolkits

What they are
Click-iT® Plus Alexa Fluor® Picolyl Azide Toolkits contain everything you need to perform copper-catalyzed click reactions with compounds that are sensitive to copper, while retaining all of the benefits of the standard azide/alkyne click reaction. Unlike the original Click-iT® azides, picolyl azide–based click reactions minimize the free copper in the click reaction, thus protecting proteins (e.g., GFP, RPE), nucleic acids (e.g., RNA, oligos), and even small molecules (e.g., phalloidin) against undesirable copper side reactions.

What they offer

• Flexibility—optimize your own Click–iT® reaction for the detection or labeling of any alkyne-containing biomolecule in vitro or in cells and tissue samples
• Brightness and sensitivity—superior fluorescent Alexa Fluor® dyes combined with the efficiency of picolyl azide
• Convenience—kits contain all the buffers and reagents needed to create the optimal click reaction

How they work
The Alexa Fluor® picolyl azide incorporates a copper-chelating group to concentrate copper at the reaction site. The copper protectant limits the concentration of free copper ions, accelerates the cycloaddition reaction, and acts as a nontoxic reducing agent to limit further reduction of copper, thus limiting the generation of reactive oxygen species (ROS). By adjusting the ratio of copper and copper protectant, one can control the amount of free copper in the click reaction and optimize it for the sample.

• Learn more about click chemistry and Click-iT® products
• Download the protocol for Click-iT® Plus Alexa Fluor® Picolyl Azide Toolkits

Sensitivity of biomolecules to copper. The effects of low, medium, and high free copper concentrations on various molecules' function and structure are shown. Green bars represent copper concentrations where the molecule functions normally; yellow and red bars represent the copper concentrations where the molecule function and/or structure is damaged or completely inhibited by copper. With the Click-iT® Plus toolkit, one can adjust the ratio of copper and copper protectant and control the amount of free copper in the click reaction, thereby optimizing it for the sample.

Bright and photostable Alexa Fluor® reference standards

Nonreactive form of Alexa Fluor® carboxylic acids

What it is
Alexa Fluor® dyes are bright, photostable, and water-soluble dyes that can be used for a broad range of applications, including fluorescence microscopy, flow cytometry, fluorescence correlation spectroscopy, and spectrofluorometry. We now offer nonreactive Alexa Fluor® carboxylic acids that can be used as reference standards or for directly labeling molecules.

What they offer

• Brightness and photostability—compared to traditional dyes, Alexa Fluor® dyes are brighter and more photostable
• pH resistance—fluorescence of the dyes is unchanged between pH 4 and 10
  
• Efficiency—the Alexa Fluor® structures allow a higher degree of labeling without intramolecular quenching
  

How they work

Our Alexa Fluor® carboxylic acids are nonreactive forms of the Alexa Fluor® dyes that can be used as reference standards for dye conjugates. Additionally, these carboxylic acid derivatives can be converted to amine-reactive esters using standard chemical techniques, or be coupled to hydrazines, hydroxylamines, and amines in aqueous solution using water-soluble carbodiimides such as EDAC.

• Learn more about the Alexa Fluor® dye series
• Learn more about all Alexa Fluor® conjugates and products

Alexa Fluor® carboxylic acid for use as a reference standard.

Improve phosphoprotein detection using the Attune® Acoustic Focusing Cytometer

The study of mitogen-activated protein kinase (MAPK) is important for investigating diseases such as cancer. MAPK signaling cascades play important roles in critical decision processes within the cell, including cellular responses to environmental stimuli and disease progression.

Multiparameter flow cytometry is a valuable tool for dissecting signaling pathways in cell populations using intracellular staining with fluorescent antibodies against phosphorylation site–specific proteins. While there are reagents and techniques available for phosphoprotein-specific detection, the signals that result from these are usually dim and difficult to distinguish. Advancements in instrumentation using acoustic focusing—the Attune® Acoustic Focusing Cytometer—enable better detection of dim signals than by conventional hydrodynamic focusing. The Attune® cytometer employs high-frequency sound waves to maintain a tightly focused sample stream, allowing greater precision at the laser interrogation point. Using the High Sensitive transit time setting to slow the sample stream allows longer laser interrogation time, which increases the sensitivity of detection.

We used three phosphoproteins, Akt, Erk1/2, and p38, to demonstrate an ideal research application for the Attune® cytometer, which enables the detection of dim signals through highly sensitive and precise data-gathering capabilities.

• Download the application note: Detection of phosphoproteins using the Attune Acoustic Focusing Cytometer
  
• Learn more about the Attune® Acoustic Focusing Cytometer

Comparison of High Sensitive and Standard transit times (using 25 μL/min sample injection rate) on the Attune® Acoustic Focusing Cytometer to the Low Flow Rate (12 μL/min) of the BD™ LSR II and BD FACSCalibur™ instruments, using Jurkat cells treated with anisomycin and stained with p38 Alexa Fluor® 488 direct conjugate. Purple traces represent untreated, p38 Alexa Fluor® 488–stained Jurkat cells; blue traces represent anisomycin-treated, p38 Alexa Fluor® 488–stained Jurkat cells. The Attune® Acoustic Focusing Cytometer demonstrates improved separation of low-expressed proteins using the High Sensitive mode, compared to the instruments using conventional hydrodynamic focusing. (A) High Sensitive, 25 μL/min, showing unstained, untreated Jurkat cells for reference (red trace), SI = 3.1. (B) High Sensitive, 25 μL/min (unstained cells not shown), SI = 3.1. (C) Standard, 25 μL/min (unstained cells not shown), SI = 2.7. (D) BD FACSCalibur™ 12 μL/min (Low Flow Rate), (unstained cells not shown), SI = 3.1. (E) BD™ LSR II 12 μL/min (Low Flow Rate), (unstained cells not shown), SI = 3.2.

Multiple choices for multiple applications

Anti-GFP antibodies

Expression of the intrinsically fluorescent green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a well-established method for visualizing specific intracellular proteins or cellular structures (e.g., mitochondria, nuclei, cytoskeleton) as well as for monitoring gene expression and other physiological processes (see references). In some instances, the fluorescent signal from GFP can be compromised (low expression) or destroyed (exposure to fixatives or paraffin embedding). In such cases, the GFP molecule may still be intact and can be detected with antibodies against the protein.

With over 400 citations, our unconjugated anti-GFP polyclonal antibodies have been the standard of a proven performer. Our anti-GFP antibodies are generated via isolation of highly purified full-length, native A. victoria protein. After inoculation of GFP into the rabbit host, the rabbit serum is purified and tested for reactivity against both native and denatured forms of GFP.

The GFP Rabbit Serum Polyclonal Antibody, which contains a highly specific antibody against GFP, is provided in complete rabbit serum. For applications requiring optimal performance, the IgG fraction is purified from the serum by ion exchange chromatography to remove nonspecific immunoglobulins. The resulting product, the GFP Rabbit IgG Polyclonal Antibody Fraction, contains only the IgG fraction from the rabbit serum raised against GFP.

The unconjugated anti-GFP polyclonal antibodies have been validated for use in the following applications:

• Immunohistochemistry (IHC)
• Immunoprecipitation (IPP)
• Western analysis
• Immunocytochemistry (ICC)
• ELISA

Learn more about:

• All of the available anti-GFP antibodies
• Citations for GFP Rabbit IgG Polyclonal Antibody Fraction
• Citations for GFP Rabbit Serum Polyclonal Antibody

Detection of GFP in transduced cells. GFP Rabbit IgG Polyclonal Antibody Fraction was labeled with Alexa Fluor® 488 dye. U2OS cells were transduced with CellLight® Mitochondria-GFP, BacMam 2.0. After an overnight incubation the cells were fixed in formaldehyde, permeabilized, and incubated with Alexa Fluor® 488–conjugated GFP antibody (green fluorescence; mitochondria). Cells were then stained with Texas Red®-X phalloidin (red fluorescence; cytoskeleton) and Hoechst 33342 (blue fluorescence; nuclei).

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.
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