ProbesOnline - June, 2011

  Read the latest issue of the ProbesOnline E-Newsletter.

In This Issue

FEATURED NEW PRODUCTS

The Tali™ Image-Based Cytometer Analyze and Visualize Cells Right at Your Bench — the Tali™ Image-Based Cytometer
   
Trial-size Antibody Conjugates for the Violet Laser Optimize Your Flow Cytometry Panel — Trial-Size Antibody Conjugates for the Violet Laser
   
ABfinity™ Recombinant Rabbit Monoclonal Antibodies Study HIF-1α With New Recombinant Antibodies — ABfinity™ Recombinant Rabbit Monoclonal Antibodies
   
Human IL-23 Heterodimer ELISA Kit Measure IL-23 Cytokine Levels — Human IL-23 Heterodimer ELISA Kit

NEW APPLICATIONS

 One-step Protocol for Antibody Labeling One-Step Protocol for Antibody Labeling
   
Enhancing GFP Detection in Neurons Enhancing GFP Detection in Neurons

PROVEN PERFORMERS

Weak signal? A case for TSA Weak Signal? A Case for TSA

GET CONNECTED

Molecular Probes® Social Media Alexa Fluor® World!
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UPCOMING MEETINGS

International Society for Stem Cell Research (ISSCR)
June 15–18
Metro Toronto Convention Centre
Toronto, Ontario, Canada
Booth #626

Southeast Flow Cytometry Interest Group
June 23–24
Georgia Center for Continuing Education
Athens, GA

Western New York Flow Users Group
July 13
University of Rochester Medical Center
Rochester, NY

OTHER PUBLICATIONS

BioProbes 65NEW! BioProbes® 65

Molecular Probes Handbook
The Molecular Probes® Handbook
11th Edition
  Order on Amazon.com

FEATURED NEW PRODUCTS


Analyze and Visualize Cells Right at Your Bench — the Tali™ Image-Based Cytometer

what it is
The Tali™ Image-Based Cytometer is a three-channel (bright-field, green and red fluorescence) benchtop assay platform that uses state-of-the-art optics, an intuitive user interface, and image analysis to perform suspension cell–based assays for GFP and RFP expression, apoptosis, cell viability, and cell counting.



what it offers

  • Quantify fluorescent protein expression
  • Analyze cell populations and cellular events (e.g., apoptosis by annexin V staining)
  • Get quantitative results with visual confirmation

how it works
Three-parameter population analysis using the Tali™ Image-Based Cytometer can be completed in approximately 1 minute using 25 µL of sample. The Tali™ cytometer performs analyses by simultaneously capturing a series of bright-field and fluorescent images and then using sophisticated digital image analysis algorithms to determine the total and fluorescent cell counts and calculate their concentrations. The Tali™ cytometer provides the option to save the assay data and to export the data as a report.

Apoptosis assay with the TaliTM cytometer

Apoptosis assay with the Tali™ cytometer
.

The Tali™ Apoptosis Kit provides Alexa Fluor® 488 annexin V and propidium iodide to assess cell viability and cell health in a population of suspension cells.

Optimize Your Flow Cytometry Panel — Trial-Size Antibody Conjugates for the Violet Laser

what they are
Molecular Probes® flow cytometry antibodies in a  trial size provide a convenient method for evaluating antibodies for your flow cytometry panel. These 405 nm–excitable fluorescent conjugates are designed to maximize use of the violet laser.



what they offer

  • 25-test size ideal for panel optimization
  • Available conjugates include Pacific Blue™ and Pacific Orange™ dyes, and Qdot® nanocrystals
  • Stocked in Supply Centers upon request

how they work
Our most popular flow cytometry antibody conjugates for the violet laser are now available in a 25-test trial size. This convenient size allows you to easily and affordably test the compatibility of these markers and fluorescent conjugates to optimize or expand your current immunophenotyping panels.


Multicolor analysis of CD3-positive and CD4-positive cell populations using Qdot® primary antibody conjugates.

Human PBLs were stained with Qdot® 605 anti-CD4 and Qdot® 655 anti-CD3 antibodies. Lymphocytes were analyzed for fluorescence using violet diode laser excitation and a 603/48 emission filter for the anti-CD4 Qdot® 605 conjugate, and 488 nm laser excitation and a 640LP emission filter for the anti-CD3 Qdot® 655 conjugate. Samples were run on the Attune® Acoustic Focusing Cytometer.

Human

Marker Conjugate
Clone
Quantity Cat. No.  
CD3Qdot® 655S4.125 testsQ10484Order Now
Pacific Blue™ dyeS4.125 testsMHCD0328TROrder Now
Pacific Orange™ dyeUCHT125 testsCD0330TROrder Now
CD4Qdot® 605S3.525 testsQ10480Order Now
Qdot® 655S3.525 testsQ10482Order Now
Qdot® 705S3.525 testsQ10485Order Now
Pacific Blue™ dyeS3.525 testsMHCD0428TROrder Now
Pacific Orange™ dyeS3.525 testsMHCD0430TROrder Now
CD8Qdot® 6053B525 testsQ10481Order Now
Qdot® 7053B525 testsQ10483Order Now
Pacific Blue™ dye3B525 testsMHCD0828TROrder Now
Pacific Orange™ dye3B525 testsMHCD0830TROrder Now
CD14Pacific Blue™ dyeTüK425 testsMHCD1428TROrder Now
Pacific Orange™ dyeTüK425 testsMHCD1430TROrder Now
CD15Pacific Orange™ dyeVIMC625 testsMHCD1530TROrder Now
CD27Qdot® 655CLB-27/125 testsQ10486Order Now
CD45Pacific Blue™ dyeHI3025 testsMHCD4528TROrder Now
Pacific Orange™ dyeHI3025 testsMHCD4530TROrder Now
CD95Pacific Blue™ dyeDX225 testsMHCD9528TROrder Now

Mouse

Marker Conjugate
Clone
Quantity Cat. No.  
CD4Pacific Blue™ dyeRM4-525 testsMCD0428TROrder Now
CD8Pacific Blue™ dye5H1025 testsMCD0828TROrder Now
Pacific Orange™ dye5H1025 testsMCD0830TROrder Now
CD45RPacific Orange™ dyeRA3-6B225 testsRM2630TROrder Now

Study HIF-1α With New Recombinant Antibodies — ABfinity™ Recombinant Rabbit Monoclonal Antibodies

what they are
Hypoxia-inducible factor (HIF-1) is a transcription factor complex made up of alpha and beta subunits. Under hypoxic conditions, HIF-1α localizes to the nucleus and binds with HIF-1β to regulate vascular endothelial growth factor (VEGF). In certain cancers, this activity can lead to tumor progression, making HIF-1α a potential target for therapeutics. 



what it offers

  • Recombinant antibodies enable consistent results
  • New antibodies released every month

 

how they work
ABfinity™ recombinant rabbit monoclonal antibodies help ensure consistent antibody performance lot after lot, so you don’t have to revalidate dilutions for your experiments when you order more. HIF-1α antibodies are available as ABfinity™ Recombinant Rabbit Monoclonal and Recombinant Oligoclonal antibody preparations, and are validated in western blotting and ELISA applications.


Western blot analysis of a whole cell extract using HIF1-α ABfinity™ Recombinant Rabbit Monoclonal Antibody.


30 µg of protein from HEK cells was separated by SDS-PAGE, followed by transfer onto nitrocellulose. The blot was blocked and then incubated with HIF-1α ABfinity™ Recombinant Rabbit Monoclonal Antibody at 0.1 µg/mL for 2 hr. A goat anti-rabbit HRP conjugate was used at 1:5,000 as the secondary antibody, and the blot was developed using chemiluminescence (ECL). The expected size of HIF-1α is ~93 kDa.

Measure IL-23 Cytokine Levels — Human IL-23 Heterodimer ELISA Kit

what it is
The Invitrogen™ Human IL-23 ELISA Kit allows accurate quantitation of IL-23 in serum or cell culture supernatant samples. IL-23 is a heterodimeric cytokine that plays a major role in survival and expansion of Th17 cells. IL-23 has a role in the development of both autoimmune and inflammatory disorders.



what it offers

  • Validated in relevant cell models, THP-1 induced with either LPS or SAA-1
  • Specificity—no cross-reactivity with 25 relevant human markers
  • Sensitivity—less than 2 pg/mL

how it works
The Human IL-23 ELISA Kit is a conventional sandwich ELISA assay that is completed after 4 hours of incubation time. The 96-well plates are precoated with IL-23 antibody. Samples are incubated, followed by several sequential incubations, and quantitative results are obtained using a microplate reader.

Human IL-23 ELISA Kit Protocol

Human IL-23 ELISA Kit protocol.
Product Quantity Cat. No.  
Human IL-23 Heterodimer ELISA Kit96 testsKHC0231Order Now

NEW APPLICATIONS


One-Step Protocol for Antibody Labeling

With names like EasyLink (Abcam) and Lightning-Link™ (Innova Biosciences), one-step antibody labeling kits from other companies promise quick protein labeling and maximum yields—up to 100%. To test how our kits compared, we labeled secondary antibodies using three of our most popular protein labeling kits—Alexa Fluor® Protein Labeling Kits, Alexa Fluor® Monoclonal Antibody Labeling Kits, and the SAIVI™ Alexa Fluor® Antibody Labeling Kit—in addition to the EasyLink and Lightning-Link™ kits. For each kit, we followed either: 1) the standard protocol, or 2) with the Molecular Probes® kits, a simplified protocol in which the final purification step was eliminated.

All three Molecular Probes® protein labeling kits produced fluorescent conjugates that effectively stained cells, even without the column purification step. Furthermore, we found that the addition of Image-iT® FX Signal Enhancer to cells prior to staining with the labeled conju­gate reduced the slight background fluorescence from free dye, producing results that were nearly indistinguishable from those obtained with a column-purified conjugate. More importantly, we found that, even without the column puri­fication step, the Molecular Probes® protein labeling kits produced fluorescent conjugates that were far superior to those produced with the other one-step labeling kits, in terms of signal strength and background fluo­rescence. Thus, with this new simplified workflow, the Molecular Probes® protein labeling kits offer one-step labeling convenience with high yields and bright results.

We offer a wide selection of protein labeling kits for covalently labeling 20 μg to 3 mg of protein with a range of Alexa Fluor® dyes, as well as with biotin and several classic fluorophores including fluorescein, Oregon Green® 488, and Texas Red® dyes. 

 

 Antibody Labeling
 

Signal-to-noise ratios for immunofluorescence staining with protein conjugates prepared using different labeling kits.
Mouse anti–human IgG labeling of HEp-2 cells on prefixed test slides (INOVA Diagnostics, Inc.) was detected with green-fluorescent goat anti–mouse IgG secondary antibodies prepared using the indicated labeling kits and protocols.
 

 

Enhancing GFP Detection in Neurons

Green Fluorescent Protein (GFP) has been used extensively in biological research to tag, localize, and purify proteins, and to study their functionality. Antibodies that can specifically detect GFP are useful tools for a variety of applications. Recent data from Dr. James I. Nagy and Dr. Mark Fry at the University of Manitoba show that the anti-GFP ABfinity™ rabbit monoclonal antibody can be used to visualize neurons in fixed tissue with high sensitivity.

Dr. Nagy and colleagues demonstrated the sensitivity of Anti-GFP ABfinity™ Rabbit Recombinant Monoclonal Antibody using tyrosine hydroxylase (TH)–GFP transgenic mice. These mice were generated to express GFP under the control of the TH gene promoter in the majority of midbrain dopamine neurons, and are useful for the study of the physiology and pathogenesis of dopamine neurons [1]. Cryosections from the substantia nigra and cortex were fixed in 4% formaldehyde, and GFP signal was enhanced using anti-GFP ABfinity™ antibody as the primary antibody and Alexa Fluor® 594 goat anti–rabbit IgG as the secondary antibody. Results showed that the signal from endogenous GFP expression was enhanced by the anti-GFP antibody, and this enhancement was not obtained using an anti-GFP antibody from a leading competitor.

The highly sensitive anti-GFP ABfinity™ antibody allows detailed visualization of neuronal structures such as cell bodies, axons, and dendrites. This antibody is also suitable for other applications, including western blots, immunoprecipitation, ELISA, flow cytometry, and fluorescent imaging.

1. Matsushita N, Okada H, Yasoshima Y et al. (2002) J Neurochem 82:295–304.

 

 Antibody Labeling
 

Visualizing neurons in transgenic mice.
Fixed cryosections of substantia nigra of TH-GFP transgenic mice. With the use of Anti-GFP ABfinity™ Rabbit Recombinant Monoclonal Antibody, the dendrites and axons are revealed, which is not possible with endogenous expression or with a competitor's antibody.

 

 

PROVEN PERFORMERS


Weak Signal? A Case for TSA

  • Ultrasensitive peroxidase-mediated signal amplification system
  • Immunohistochemical detection of low-abundance targets
  • Minimized diffusion-related loss of signal localization
  • Ideal solution for multiplex studies

Powerful Signal Amplification
Tyramide signal amplification (TSA) is an enzyme-mediated detection method that uses the catalytic activity of horseradish peroxidase (HRP) to generate high-density labeling of a target protein or nucleic acid sequence in situ. The Molecular Probes® TSA Detection Kits combine the versatile and powerful TSA technology with our high-performance Alexa Fluor® dyes, as well as our classic dyes like Oregon Green® and Pacific Blue™. The technology can also be used with colorimetric detection systems using our TSA biotin or DNP kits.

Applications of TSA Technology
For low-abundance targets, poor antibodies, or other reasons for a low signal, TSA technology has been proven in a number of systems and applications. The signal amplification derived from multiple tyramide substrates per peroxidase label translates to ultrasensitive detection of low-abundance targets and the use of smaller amounts of antibodies and hybridization probes. The increased sensitivity afforded by TSA technology can be critically important for detection of short oligonucleotide probes and low-abundance mRNAs by fluorescence in situ hybridization (FISH). Optimal probe concentrations are typically 2- to 10-fold lower for TSA-detected FISH than for conventional immunocytochemical detection procedures.



Signal amplification in a zebrafish retina
 
Signal amplification in a zebrafish retina.
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. The sample was then incubated with a mouse monoclonal FRet 6 antibody and visualized with Alexa Fluor® 647 goat anti–mouse IgG, which is pseudocolored magenta. Finally, the nuclei were counterstained with SYTOX® Orange Nucleic Acid Stain.


  1. Merzlyak EM, Goedhart J, Shcherbo D et al. (2007) Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat Methods 4(7):555–557.

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The View


 
Multiplex imaging of apoptosis.
U2OS cells were treated with 30 μM etoposide for 18 hr to induce apoptosis. The treated cells were stained first with 7.5 μM CellEvent™ Caspase-3/7 Green Detection Reagent (Cat. No. C10423, green fluorescence) to detect apoptosis, and Hoechst 33342 nucleic acid stain (Cat. No. H3570, blue fluorescence) to label nuclei, and then with 150 nM MitoTracker® Deep Red FM (Cat. No. M22426, pink fluorescence) to label mitochondria. Following fixation and permeabilization, actin was labeled with Alexa Fluor® 546 phalloidin (Cat. No. A22283, orange fluorescence). Image contributed by Michelle Yan, Life Technologies Corporation.

 

Product Quantity Cat. No.  
CellEvent™ Caspase-3/7 Green Detection Reagent100 µLC10423Order Now
MitoTracker® Deep Red FM20 x 50 µgM22426Order Now
Hoechst 33342100 µLH3570Order Now
Alexa Fluor® 546 Phalloidin300 unitsA22283Order Now

Buzzworthy

15-μm-Sized Single-Cellular-Level and Cell-Manipulatable Microplasma Jet in Cancer Therapies

Kim JY, Wei Y, Li J, Kim SO (2010) Biosens Bioelectron 26:555–559.

In a recent publication, Kim et al. describe an innovative new approach for inducing apoptosis in cancer cells. The authors used a "microplasma jet" device to precisely deliver atmospheric-pressure plasma—composed of ionized charged particles, free electrons, and radicals—into individual cultured murine melanoma tumor cells. Using the Click-iT® TUNEL Alexa Fluor® 488 Imaging Assay for Apoptosis, which employs click chemistry for the detection of DNA fragmentation associated with apoptosis, the authors observed the induction of apoptosis in a dose-dependent manner. Furthermore, the tumor cells were more sensitive to plasma treatment than murine fibroblast cells. The authors speculate that this novel microplasma jet technology can be used to support targeted cancer therapy approaches with improved precision.



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