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In This Issue
|Tools for Mitochondrial Research — New Mitochondrial Antibodies|
|Sensitive and Accurate Nucleic Acid Quantitation — Introducing the Qubit® 2.0 Fluorometer and the Qubit® Fluorometric Platform|
|Specific Quantitation of Growth Hormone — Rat Growth Hormone ELISA Kit|
|Multiplex Western Blot Detection|
|Method for Fluorescent Labeling of IgM Antibodies|
Buzzworthy — GlcNAcylation Plays an Essential Role in Breast Cancer Metastasis
The View — Visualizing Actin and Tubulin in Mouse Embryo Fibroblasts
- What's New — Condense Hours of Searching and Reading into 5 Minutes a Day
- On the Web — Product Selection Guides and Research Tools
- Explore BioProbes 63
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FEATURED NEW PRODUCTS
what they are
Invitrogen offers a diverse array of products for studying mitochondrial biology and for determining the role of mitochondria in specific disease states or pathways. These tools include antibodies, Molecular Probes® dyes, and assays for mitochondrial protein expression. New antibodies are available for the detection of specific mitochondrial proteins, including GAPDH, HSDL2, DCXR, and SOD2.
what they offer
- Superior specificity for mitochondrial proteins
- Validated for multiple applications and species specificities
- Part of a wide-ranging portfolio of antibodies for mitochondrial research
how they work
Aside from energy production, mitochondria also help to regulate a variety of cellular processes and functions. Invitrogen™ mitochondrial antibodies can be used to study the role of mitochondria in various cellular pathways, as well as their role in mitochondria-related diseases such as Parkinson’s, Alzheimer’s, Huntington’s, Friedreich's Ataxia (FRDA), and various cancers. Our antibodies can be used reliably in multiple applications, including western blots, immunocytochemistry, immunohistochemistry, immunoprecipitation, and ELISA.
Immunocytochemistry of fibroblast cells labeled with L-xylulose reductase (DCXR) monoclonal antibody.
Fibroblast cells grown on slides were paraformaldehyde-fixed (4%, 20 min) and permeabilized with Triton® X-100 (0.1%, 15 min). Primary antibody: L-xylulose reductase (DCXR) monoclonal antibody. Secondary antibody: Alexa Fluor® 488 goat anti–mouse IgG (H+L) at a 1:1,000 dilution for 1 hr. 10% goat serum was used for all blocking steps. DAPI was used to stain the nuclei (blue).
||WB, IF/ICC, IP
||WB, IP, IF/ICC, In-Cell ELISA
||B, Hu, Ms, Rt
||IF/ICC, IP, In-Cell ELISA
||WB, IP, IF/ICC, In-Cell ELISA
||IP, IF/ICC, IHC, In-Cell ELISA
||B, Hu, Ms, Rt
† Reactive species: B = bovine; Hu = human; Ms = mouse; Rt = rat.
The Qubit® Fluorometric Platform combines the newly redeveloped Qubit® 2.0 Fluorometer with the Qubit™ assays for nucleic acid quantitation. The instrument and assays have been seamlessly integrated to generate a quantitation platform that is far more sensitive and accurate than UV absorbance. Qubit™ assays (previously known as Quant-iT™ assays) are compatible with both Qubit® 1.0 and Qubit® 2.0 Fluorometers.
what it offers
- Larger LCD color touch screen
- Automated data logging and USB port for efficient data management
- Displays a standard curve after the calibration
- Step-by-step workflow navigation
As a result of customer feedback, the next-generation Qubit® 2.0 Fluorometer has an improved user interface and data storage and transfer mechanisms, making the instrument more intuitive and easy to use. The Qubit™ assays, which utilize the proven performance of Molecular Probes® fluorescent dyes, complete the seamless integration of the platform, resulting in the highest level of accuracy and specificity available for nucleic acid quantitation.
- Learn More About Qubit® Fluorometric Quantitation
Selectivity of the Qubit™ assays compared to UV spectroscopy.
Triplicate samples containing lambda DNA (10 ng/μL) and varying amounts of E. coli ribosomal RNA (0–100 ng/μL) were assayed using Qubit™ DNA BR and Qubit™ RNA BR assays according to kit protocols. The same samples were subsequently measured in triplicate using a NanoDrop® ND-1000 Spectrophotometer, and single measurements were made using a PerkinElmer Lambda 35 Spectrophotometer. The red and orange trendlines indicate the actual concentrations of DNA and RNA, respectively, in the starting samples. The data indicate that UV analysis cannot distinguish between DNA and RNA.
what it is
The Rat Growth Hormone ELISA Kit detects and quantifies growth hormone (GH) in rat serum, plasma, or supernatant samples. GH has diverse effects on cell growth, differentiation, and metabolism. The kit cross-reacts with mouse.
what it offers
- Specificity—sandwich ELISA uses 2 antibodies against growth hormone for greater specificity
- Ease of use—simple protocol and step-by-step instructions
- Consistency—reproducible results
how it works
The sandwich ELISA utilizes a simple protocol to detect growth hormone. Incubate your diluted samples or standard in the antibody-coated 96-well plate. Growth hormone in the sample will bind to the plate, and everything else is washed away. A second, biotinylated antibody is added and binds to a different site of growth hormone. After washing, streptavidin-HRP is added and binds to the biotinylated antibody. After washing to remove all unbound enzyme, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of the color is directly proportional to the concentration of rat growth hormone present in the sample.
- Learn More About ELISA Kits
Growth Hormone ELISA Kit protocol.
To visualize more than one protein of interest on a western blot, traditional detection technology requires preparing multiple blots, or sequential stripping and reprobing of a single blot. Now, simply by integrating WesternDot™ detection technology into your current WesternBreeze® chemiluminescent western blot workflow, two proteins can be visualized simultaneously on the same blot, with no additional solutions or steps.
For multiplexed western blot detection, a single blot is coincubated with both a mouse and a rabbit primary antibody, then with a goat anti-mouse or goat anti-rabbit alkaline phosphatase secondary antibody and the alternate biotinylated secondary antibody, followed by incubation with the streptavidin–Qdot® 625 conjugate. The Qdot® signal can then be imaged before, during, or after incubation with the WesternBreeze® CDP-Star® detection reagent (see figure). The remarkable photostability of the Qdot® nanocrystal allows the blots to be dried and re-imaged days and even months later, and the signal is not diminished by the WesternBreeze® enzymatic reaction.
|WesternDot™ 625 Goat Anti-Mouse Western Blot Kit||1 kit||W10132|
|WesternDot™ 625 Goat Anti-Rabbit Western Blot Kit||1 kit||W10142|
|WesternBreeze® Chemiluminescent Kit–Anti-Mouse||1 kit||WB7104|
|WesternBreeze® Chemiluminescent Kit–Anti-Rabbit||1 kit||WB7106|
|WesternBreeze® Chromogenic Kit–Anti-Mouse||1 kit||WB7103|
|WesternBreeze® Chromogenic Kit–Anti-Rabbit||1 kit||WB7105|
|Novex® AP Chemiluminescent Substrate (CDP-Star®)||100 mL||WP20002|
|Novex® AP Chemiluminescent Substrate Enhancer (Nitro Block II™)||5 mL||WP20003|
|Novex® ECL Chemiluminescent Substrate Reagent Kit||2 x 125 mL||WP20005|
Dye-conjugated IgM antibodies offer a method of live-cell labeling for the identification and characterization of pluripotent stem cells, including embryonic and induced pluripotent stem cells. IgM antibodies can be conveniently labeled with our superior amine-reactive Alexa Fluor® dyes (Alexa Fluor® NHS ester and SDP ester). Their structural complexity and sensitivity to pH and other physical conditions require modification of protocols optimized for IgG-class antibodies. A protocol for IgM labeling has been optimized in our laboratory with Alexa Fluor® amine-reactive dyes, which react with nonprotonated aliphatic amine groups (the α-terminus and lysine ε-amino groups).
- View the Protocol for IgM Labeling
|Detecting stem cell surface markers using fluorescently labeled IgM antibodies. Tra-1-81 antibodies were labeled using (A) Alexa Fluor® 488 SDP ester, (B) Alexa Fluor® 594 NHS ester, and (C) Alexa Fluor® 647 NHS ester. H9 human embryonic stem cells on a feeder layer of irradiated murine embryonic fibroblasts were cultured on hESC medium containing 20% KSR and 4 ng/mL bFGF. Cells were stained with dye-conjugated antibodies at 5 µg/mL, and images were collected on an Axiovert fluorescence microscope at 10x magnification.|
|Alexa Fluor® 488 NHS Ester
|Alexa Fluor® 488 SDP Ester
|Alexa Fluor® 594 NHS Ester
|Alexa Fluor® 647 NHS Ester
GlcNAcylation Plays an Essential Role in Breast Cancer Metastasis.
Gu Y, Mi W, Ge Y et al. (2010) Cancer Res 70:6344–6351.
GlcNAcylation is a posttranslational modification (O-GlcNAc moiety linked to the side chain hydroxyl of serine or threonine) found on numerous cytoplasmic and nuclear proteins. This modification regulates both normal and disease-related biological processes, and several studies have shown that GlcNAcylation acts as a cellular regulator of growth and division. In a recent publication, Gu and colleagues investigated the role of GlcNAcylation in breast cancer metastasis. Immunohistochemical staining showed that global GlcNAcylation levels were significantly elevated in human breast tumor tissues compared to adjacent nondiseased tissues. 4T1 mouse tumor cells were used in subsequent experiments to (1) metabolically label O-GlcNAc moieties (using the Click-iT® GlcNAz Metabolic Glycoprotein Labeling Reagent) and (2) to silence the O-GlcNAc transferase (OGT) gene (responsible for removing O-GlcNAc moieties) so that the role of GlcNAcylation of E-cadherin could be observed. The authors discovered that GlcNAcylation decreases cell surface E-cadherin, which, in turn, enhances cell migration and metastasis. These findings highlight GlcNAcylation as a potential target for future therapy efforts.
||Visualizing actin and tubulin in mouse embryo fibroblasts. Albino Swiss mouse embryo fibroblast cells (3T3) were labeled with Alexa Fluor® 546 phalloidin (F-actin; orange), α-tubulin mouse monoclonal antibody Alexa Fluor® 488 conjugate (tubulin; green), and TO-PRO®-3 iodide (nucleus; red).
|Product Selection Guides
Our new selection guides provide free access to research protocols, product selection and design tools, pathway maps, and gene information to support your research needs. Each guide is simple and easy to use and contains scientific information you can rely on to successfully plan and complete your experiments.
The Molecular Probes® Handbook
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