ProbesOnline™ March 2014

In this issue

FEATURED NEW PRODUCTS

Tyramide Signaling Amplification Kits   Detect dim or low-abundance targets—New Tyramide Signaling Amplification Kits
     
chemokine ligands by flow cytometry   Study chemokine ligands by flow cytometry—New hamster monoclonal anti–human/mouse/rat CCL2 (MCP-1) antibody conjugates
     
Antibody Conjugate Purification Kits   Simple and convenient antibody conjugate purification—Molecular Probes® Antibody Conjugate Purification Kits
     
Click-iT® Plus OPP Protein Synthesis Assays   Rapid detection of new protein synthesis in complete media—Click-iT® Plus OPP Protein Synthesis Assays
     
WesternDot® 625   Simplify your western blot detection—WesternDot® 625 secondary antibody conjugates

NEW APPLICATIONS

FluoroMyelin™ Red FluoroMyelin™ Red is a bright, photostable and non-toxic fluorescent stain for live imaging of myelin

PROVEN PERFORMERS

Qtracker® cell labeling kits   Qtracker® cell labeling kits for stem cell research

OTHER PUBLICATIONS

BioProbes® Journal of Cell Biology Applications

The Molecular Probes® Handbook

 UPCOMING EVENTS

Association of Biomolecular Resource Facilities (ABRF) 2014
March 22–25
Albuquerque Convention Center
Albuquerque, New Mexico, USA

Southern California Flow Cytometry Association 2014 SoCal Flow Summit
March 24–25
Beckman Center, UC Irvine
Irvine, California, USA

Analytica
April 1–4
Messe München International
Munich, Germany

American Association for Cancer Research (AACR) Annual Meeting
April 5–9
San Diego Convention Center
San Diego, California, USA

School on Diagnostic Assessment of Phenotype and Function in Primary Immunodeficiencies
April 10
Hilton Baltimore
Baltimore, Maryland, USA

FEATURED NEW PRODUCTS

Detect dim or low-abundance targets

New Tyramide Signaling Amplification Kits

What they are
For ultimate detection sensitivity, tyramide-based amplification techniques are widely used in fluorescent immunodetection and FISH procedures to visualize targets with dim or low expression levels. This tyramide signaling amplification (TSA) system can be used with both chromogenic and fluorescent signals on slide-based assays. Our new stand-alone Alexa Fluor® and biotin–tyramide conjugates are designed to work with any HRP-conjugated secondary and come with the necessary amplification buffer. Also available are complete TSA kits for more common antibody species.


What they offer

  • Detection of low-abundance targets—TSA-amplified signals can be 100-fold stronger than those from traditional immunodetection techniques
  • Increased versatility—these stand-alone conjugates can be used to perform TSA using any horseradish peroxidase (HRP)-conjugated secondary or primary antibody

How they work
TSA is an enzyme-mediated detection method that uses HRP to generate high-density labeling of a target protein or nucleic acid in situ. A tyramide conjugate is incubated in the presence of HRP (often bound to a secondary antibody), which catalyzes the binding of the tyramide (and associated label) to adjacent tyrosines for enhanced fluorescent signal (if the tyramide is conjugated to a fluorescent dye) or colorimetric detection (if the tyramide is conjugated to biotin).

In addition to the stand-alone format offered here, tyramide conjugates are also available as part of an optimized kit that includes a secondary antibody-HRP conjugate or streptavidin-HRP conjugate and other essential components. For the first-time users, we recommend use of the optimized kit. However, if the primary antibody used is not compatible with the secondary antibodies available with these kits, stand-alone tyramide conjugates (with amplification buffer) are offered that may be used as part of a customized system.

Flow cytometry identification of a chemokine ligand  


Sensitive detection of zebrafish α-tubulin.
A zebrafish cryosection was incubated with the biotin-XX conjugate of mouse monoclonal anti–α-tubulin antibody. The signal was amplified with TSA™ Kit #22, which includes HRP–streptavidin and Alexa Fluor® 488 tyramide (green). The sample was then incubated with a mouse monoclonal FRet 6 antibody and was visualized with Alexa Fluor® 647 goat anti–mouse IgG (magenta). Finally, the nuclei were counterstained with SYTOX® Orange nucleic acid stain (orange).

 


Study chemokine ligands by flow cytometry

New hamster monoclonal anti–human/mouse/rat CCL2 (MCP-1) antibody conjugates

What they are
The Molecular Probes® portfolio of over 1,600 highly specific primary antibodies for flow cytometry is expanding to include more Research Use Only (RUO) selections. FITC and phycoerythrin conjugates of hamster anti–human/mouse/rat CCL2 (MCP-1) antibody are now available, giving you more options for studying chemokine ligands.


What they offer

  • Trust—Molecular Probes® brand
  • Validation—all antibodies are validated for flow cytometry applications
  • Selection—expanded offerings of primary antibody conjugates for flow cytometry

How they work
The hamster anti–human/mouse/rat CCL2 (MCP-1) monoclonal antibody (mAb) recognizes mouse, rat, and human chemokine (C-C motif) ligand 2 (CCL2), also called monocyte chemoattractant protein 1 (MCP-1).

Flow cytometry identification of a chemokine ligand  
Flow cytometry identification of a chemokine ligand in cells.
Staining of 1-day LPS-stimulated human peripheral blood cells, with Armenian hamster IgG isotype control FITC (open histogram) or anti-CCL2 (MCP-1) FITC (shaded histogram). Cells in the monocyte gate were used for analysis.


Simple and convenient antibody conjugate purification

Molecular Probes® Antibody Conjugate Purification Kits

What they are
Molecular Probes® Antibody Conjugate Purification Kits efficiently purify antibody conjugates from unconjugated dye or biotin. Each kit contains sufficient material for multiple purifications and, in addition to antibodies, can be used to purify almost any high molecular weight protein (>40 kDa) from unconjugated labels.


What they offer

  • Fast—purified antibody conjugate ready to use in as little as 10 min
  • Easy—minimal setup time (~5–15 min)
  • Compatible—optimal for use with Alexa Fluor® and biotin labeling reagents

How they work
For 0.5–1 mg of antibody conjugate, removal of unconjugated dye or biotin is accomplished using an easy-to-assemble gravity-fed size-exclusion column containing Bio-Gel® P-30 resin. For smaller amounts of antibody (20–100 µg), spin columns are used to separate the conjugated antibody or other protein from the label. These Antibody Conjugate Purification Kits are designed to remove low molecular weight labels (<3,000 Da, e.g., Alexa Fluor® dyes) and cannot be used for removing unconjugated high molecular weight labels (>10 kDa) such as enzymes or other proteins (e.g., horseradish peroxidase or R-phycoerythrin).

Molecular Probes® Antibody Conjugate Purification Kits  
Molecular Probes® Antibody Conjugate Purification Kit.
 

Rapid detection of new protein synthesis in complete media

Click-iT® Plus OPP Protein Synthesis Assays

What they are
The Click-iT® Plus OPP Protein Synthesis Assays provide a fast, sensitive, and nonradioactive method for the detection of protein synthesis in complete media using fluorescence microscopy, high-content imaging, or flow cytometry. Assays are available with Alexa Fluor® 488, 594, or 647 dye.


What they offer

  • No media change required—works in complete, methionine-containing media
  • Multiplex-enabled—retains signal from GFP and actin binding and signal of fluorescently labeled phalloidins
  • Nonradioactive—an alternative to the traditional 35S-methionine methods
  • Works in vivo—published results demonstrate use for in vivo protein translation determination

How they work
O-propargyl-puromycin (OPP), an alkyne analog of puromycin, is fed to cultured cells in complete media and is incorporated into proteins during active protein synthesis. Addition of an Alexa Fluor® picolyl azide and the click-reaction reagents leads to a chemoselective ligation or click reaction between the picolyl azide dye and the OPP alkyne, allowing the modified proteins to be detected by image-based analysis. Unlike the original non-picolyl click azides, the picolyl azide reaction conditions are very mild such that cell morphology, the binding of fluorescently labeled phalloidins, and the fluorescent signal from GFP are preserved.

Click-iT® Plus OPP Protein Synthesis Assays  
Protein synthesis visualization in HeLa cells.
CellLight® Talin-GFP, BacMam 2.0 was used to transduce HeLa cells. After overnight incubation, the Click-iT® OPP reagent supplied in the Click-iT® Plus OPP Alexa Fluor® 647 Protein Synthesis Assay Kit was added and incubated for 30 minutes. After incubation, the cells were fixed, permeabilized, and stained with Alexa Fluor® 568 phalloidin. Nascent protein synthesis was then detected using Alexa Fluor® 647 picolyl azide. Talin-GFP fusion protein bound to cytoskeleton appears green, Alexa Fluor® 568 phalloidin bound to F-actin appears red, and nascent protein synthesis detected with Alexa Fluor® 647 picolyl azide is pseudocolored blue.

Simplify your western blot detection

WesternDot® 625 secondary antibody conjugates

What they are
Our new fluorescent WesternDot® 625 secondary antibody conjugates, powered by Qdot® VIVID® technology, allow researchers to simplify western blot detection without sacrificing performance. Users can obtain results equivalent to enhanced chemiluminescence (ECL) using standard UV light boxes and gel imagers equipped with ethidium bromide or SYBR® Green detection filters (see figure). WesternDot® secondary antibody conjugates eliminate the need for film, developer solutions, darkrooms, and enzyme optimization.


What they offer

  • Easy detection—fewer steps than ECL detection, and eliminate the need for X-ray films and developer solutions
  • Stable signal—fluorescence signal does not fade over time
  • Convenience—detect using the same UV or digital imaging systems used for gels stained with ethidium bromide or SYBR® Green dyes

How they work
Each vial contains enough reagent to stain 25 mini blots when using the recommended 1:500 dilution. WesternDot® secondary antibody conjugates are compatible with common blocking reagents such as casein and powdered milk. Converting from ECL to WesternDot® staining is straightforward, and by eliminating the need for enzymes the workflow is shortened and simplified, particularly when switching from film-based detection. To detect target proteins, simply image the blot the same way that you would image a gel stained with ethidium bromide or SYBR® Green dyes. Furthermore, unlike chemiluminescent signals, which fade over time, WesternDot® signals are extremely stable and can last for days to months.

WesternDot® 625 secondary antibody conjugates   Comparison of enhanced chemiluminescence (ECL) and WesternDot® fluorescence for western blot protein detection. Serial dilutions (10–0.02 µg) of Jurkat lysate were run on NuPAGE® Novex® 4–12% Bis-Tris precast gels  and transferred to iBlot® nitrocellulose membranes using the iBlot® Gel Transfer Device. The membranes were probed with mouse anti-GAPDH antibodies followed by either fluorescence detection using WesternDot® 625 goat anti–mouse IgG (A–F) or ECL detection using a horseradish peroxidase goat anti–mouse IgG (G). WesternDot® images were collected using multiple imagers. Filter settings for fluorescence detection on each imager are as follows: A, Qdot® 625 filter; B, 645AF20 filter; C, LPG filter; D, ethidium bromide filter; E, ethidium bromide filter; F, iPhone® 5 camera and UVP™ Mini Benchtop UV Transilluminator. The ECL image (G) was acquired using the Fujifilm® LAS-4000.

NEW APPLICATIONS

FluoroMyelin™ Red is a bright, photostable and non-toxic fluorescent stain for live imaging of myelin

Monsma PC, Brown A (2012) J Neurosci Methods 290:344–350

Myelin plays a critical role in nervous system function, facilitating the conductance of neural signals. Traditionally, visualization methods include the use of antibodies (e.g., anti–myelin basic protein) or chromogenic (transmitted-light) methods (e.g., the Loyez method or Schmued’s gold chloride technique), all of which are time consuming, requiring multiple steps over one to three days. As an alternative, Molecular Probes® FluoroMyelin™ Red fluorescent myelin stain selectively labels myelin in sections of frozen or chemically fixed tissue within 20 minutes after adding the stain [1,2].

In this article, Monsma and Brown demonstrate that FluoroMyelin™ Red stain is also effective for selectively labeling myelin in living myelinating co-cultures of Schwann cells and dorsal root ganglion neurons. In the study, they established the amount of time needed to stain the cells (2 hours), the dyes's photostability, which was found to be sufficient for performing long-term time-lapse imaging, the reversibility of the staining (an approximate half-life of 130 minutes), and the impact of the stain on long-term living co-cultures (no apparent adverse effects were observed by light microscope). The authors confirmed, using the Molecular Probes® LIVE/DEAD® Cell Viability Kit, that the stain was not toxic to cells. Additionally, they found that FluoroMyelin™ Red stain's fluorescence could be distinguished from that of mCherry red fluorescent protein using a custom bandpass epifluorescence filter set. The authors concluded that FluoroMyelin™ Red stain will be useful for selective myelin staining in living co-cultures due to its high water solubility, its ease of use, and its low impact on cell function and viability.

  1. Kanaan A, Farahani R, Douglas RM, Lamanna JC, Haddad GG (2006) Am J Physiol Regul Integr Comp Physiol 290:R1105–14.
  2. Watkins TA, Emery B, Mulinyawe S, Barres BA (2008) Neuron 60:555–69.
FluoroMyelin™ Red   Live imaging of myelinated axons in long-term myelinating co-cultures of Schwann cells and dorsal root ganglion neurons. The axons are expressing GFP-tagged neurofilament protein (green), and the myelin is stained with FluoroMyelin™ Red stain (red). The image is a maximum intensity projection obtained from z-stacks acquired by spinning disk confocal microscopy using a 60x/1.2 NA objective. Image contributed by Anthony Brown, Ph.D., Department of Neuroscience, The Ohio State University College of Medicine.

PROVEN PERFORMERS

Qtracker® cell labeling kits for stem cell research

Tracking stem cells in vivo often requires labeling of the isolated stem cells in culture with a tracking molecule that can be easily distinguished from host cells post-treatment. Qtracker® Cell Labeling Kits are designed for labeling cells grown in culture with highly fluorescent and photostable Qdot® nanocrystals. Once inside the vesicles of the stem cells, Qtracker® labels provide intense, stable fluorescence that can be traced through several generations, and the labels are not transferred to adjacent cells in a population. The fast, convenient workflow requires minimal hands-on time, and the fluorescence intensity persists through acetone fixation of tissue samples [1]. In addition, Qtracker® labeling does not affect viability, proliferation, or differentiation potential of stem cells [2-3].

  1. Muccioli M, Pate M, Omosebi O, and Benencia F (2011) J Vis Exp 52:2785.
  2. Lin S, et al. (2007) BMC Biotechnology 7:67.
  3. Rak-Raszewska A, et al. PLoS One (2012) 7:e32650.

 
Stem cell tracking with Qtracker® 655 nanocrystals.
Human umbilical vein endothelial cells (HUVEC) were stained with the Qtracker® 655 Cell Labeling Kit (magenta). The cells were then fixed, permeabilized, and stained with ReadyProbes™ NucBlue® Fixed Cell Stain (blue) and ActinGreen™ 488 Reagent (green). The figure is a maximum-intensity projection of Z-stack images collected on a Zeiss® 710 LSM confocal microscope.

DEPARTMENTS

On the web

Exosomes—the next small thing: A mini-documentary series

We asked 10 prominent scientists to share their thoughts—on camera—on the exciting field of exosome research. Based on these interviews, we produced a six-part mini-documentary series that tells the history and story of this new field and its impact on research areas such as cancer and immunology. The series also gives an overview of potential future therapeutic and diagnostic applications that may come from exosome research. Whether you’re into exosomes or not, this is a must-see!

 
Exosomes—The next small thing: A mini-documentary series
 

Imaging corner

Live-cell super-resolution imaging (SRM) of mitochondria and Golgi apparatus

Enlarge Image  
HeLa cells were transduced with CellLight® Mitochondria-GFP, BacMam 2.0 (green) and CellLight® Golgi-RFP, BacMam 2.0 (orange) and co-stained with Hoechst 33342 (blue). Structured illumination microscopy (SIM) images were acquired using the DeltaVision® OMX system (Applied Precision®, GE Healthcare). Image courtesy of Ian Clements, GE Healthcare.

Highlight from BioProbes® Journal

No better time to react: Painting biomolecules with reactive fluorophores

The poster in the center of the print version of BioProbes® 69 describes our extensive selection of reactive fluorophores for labeling antibodies, other proteins and peptides, oligonucleotides, and other molecules of interest. At a glance, you can see a wide range of fluorescent labels organized by emission color and reactivity, along with some key examples of their use in FISH, immunocytochemistry, in vivo labeling with fluorescent conjugates, and flow cytometry. Hang this poster on your lab fridge, and your labeling options will be right where you need them.

  fluorophore poster  
Reactive fluorophores poster.
This downloadable poster features our most popular fluorescent labels, listed in order of their emission wavelengths. The reactivity and typical uses of each label are also listed.
 

† What's new with the BioProbes® Journal?

We are bringing our award-winning BioProbes® articles to you sooner. We will be publishing new BioProbes® articles online every month and highlighting those articles here. That way, we can keep you up-to-date on new fluorescence technologies and cell biology applications. Check back frequently and watch BioProbes® 70 take shape!