- Read the latest issue of the ProbesOnline E-Newsletter.
WesternDot™ 625 Western Blot Kit
what it is
The WesternDot™ 625 Western Blot Kits combine the bright fluorescence properties of Qdot® 625 nanocrystals with the high-affinity streptavidin-biotin interaction to allow simple, highly sensitive detection of proteins immobilized on nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membranes. You get the sensitivity of chemiluminescence methods combined with brightness, photostability, flexibility and greater ease of processing and imaging—no x-ray film, dark rooms, or developing reagents are required.
how it works
Incorporating a standard western blotting protocol, the detection step relies on a biotinylated secondary antibody, either goat anti-rabbit (W10132) or goat anti-mouse (W10142) followed by the key component, the Qdot® 625 streptavidin conjugate (Figure 1). The extremely high extinction coefficient of the Qdot® 625 nanocrystal in the UV and blue wavelengths combined with high quantum yield and an orange/red emission (Figure 2) allow for subnanogram sensitivity of protein detection using standard UV or blue-light based detection systems. The fluorescent signal is compatible with the commonly used modes of fluorescence detection of DNA or protein gels and does not require specialized emission filters. Blots may be imaged wet or dry, and by epi- or trans-illumination.
Figure 1—Workflow diagram for the WesternDot™ 625 Western Blotting Kit.
|Figure 2—Example of results using the WesternDot™ 625 Western Blotting Kit. Total proteins (2-fold dilution series ranging from 10 μg to ~10 ng) from Jurkat cell extract were analyzed on a NuPAGE® Novex® 4–12% Bis-Tris gel and then transferred to an Immobilon™-FL PVDF membrane. Immunodetection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and endogenous “housekeeping” protein in the Jurkat cell extract, was performed with the WesternDot™ 625 Goat Anti-Mouse Western Blot Kit using a mouse monoclonal anti-GAPDH antibody (Invitrogen Cat. no. 39-8600) at 1 μg/ml. The wet membrane was imaged using an Alpha Innotech HD2 instrument with a SYPRO® Red emission filter (620 +/– 40 nm) and excitation at 302 nm transillumination with an exposure time of 300 milliseconds.|
what it offers
- sensitivity equivalent to chemiluminescence methods
- easy to use: no need to optimize ECL reagents for the best image
- wet or dry imaging in either epi- or trans-illumination mode
- hassle free: signal on the processed blot remains stable for subsequent imaging
Find out more at www.invitrogen.com/qdots.
Click-iT® EdU Microplate Assay
what it is
The Click-iT® EdU Microplate Assay (C10214) is a fluorescence-based assay for accurate, sensitive, and fast quantitation of proliferating cells through the incorporation of the novel nucleoside, EdU (5-ethynyl-2’-deoxyuridine) during nascent DNA synthesis.
how it works
The Click-iT® EdU assay provides an alternative to antibody-based bromodeoxyuridine (BrdU) cell proliferation assays. EdU detection is based on a click reaction—a copper-catalyzed covalent reaction between an azide and an alkyne. In this assay, the EdU contains the alkyne and the Click-iT® Oregon Green® 488 dye contains the azide. The small size of the fluorescent dye azide (MW <~1000) compared to an anti-BrdU antibody (MW ~150,000) allows for efficient detection of the incorporated EdU using mild conditions (see figure). The EdU signal is then amplified using horseradish peroxidase (HRP) which then reacts with Amplex® UltraRed substrate to produce a bright, red-fluorescent product (excitation/emission maxima ~568/585 nm).
Detection of the incorporated EdU with the Oregon Green® 488 azide versus incorporated BrdU with an anti-BrdU antibody.
what it offers
- higher sensitivity and broader assay range than fluorescence- or absorbance-based BrdU microplate cell proliferation assays
- the opportunity to visualize cells by fluorescence microscopy or high-throughput imaging before the signal amplification and detection steps
- stop reagent that stabilizes the fluorescent signal for up to 24 hours
- answers to your cell proliferation query in <2 hours
Visit www.invitrogen.com/edu for more information on our other Click-iT® EdU products for flow cytometry, imaging, and high-throughput imaging.
Matrix Metalloproteinase-9 (MMP-9) ELISA Kits
what is it
Invitrogen is now offering assay kits to measure human MMP-9 in serum, plasma, and cell culture supernatant samples. MMPs are proteolytic enzymes that are responsible for the degradation of extracellular matrix proteins and are involved in an array of physiological and pathological processes from development, morphogenesis, reproduction, wound healing, inflammation, angiogenesis, neurological disorders, and cancer cell invasion and metastasis.
how it works
The ELISA kit is a simple and unbiased way to quantify specific proteins. The Invitrogen Human MMP-9 Kit (KHC3061, KHC3062) is a solid-phase sandwich Enzyme Linked Immunosorbent Assay (ELISA). A highly purified antibody has been coated onto the wells of the microtiter strips provided. During the first incubation, standards of known Human MMP-9 content, controls, and unknown samples are pipetted into the coated wells. After washing, biotinylated second antibody is added, which is followed by washing and the addition of streptavidin peroxidase. This binds to the biotinylated antibody to complete the four member sandwich. After a third incubation and washing to remove all the unbound enzyme, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of Human MMP-9 present in the original specimen. Invitrogen ELISA kits come ready-to-use with all the necessary reagents including calibrated standards and controls.
what it offers
- sensitive—use small sample volume
- specific—measure the correct protein of interest
- fast—4 hours to complete
- minimal variation—consistent lot-to-lot performance
Demonstration of parallelism between natural and recombinant MMP-9. Random human serum, plasma and cell culture medium samples were serially diluted in the Standard Diluent Buffer and analyzed. The optical density of each dilution was plotted against the Human MMP-9 standard curve. Parallelism was demonstrated by the figure and indicated that the standard accurately reflects the Human MMP-9 content in natural samples.
Learn More about ELISA Kits.
Saretzki, G. et al. (2008) Stem Cells 26:455.
How do stem cells maintain a low mutational load? In order to retain their capacity to differentiate, stem cells have to maintain genetic stability over multiple generations. This is believed to occur through two mechanisms: by reducing spontaneous mutation frequencies through lowered physiological stress and increased activity and fidelity of repair processes, and by the elimination of damaged stem cells through differentiation or cell death. Reactive oxygen species (ROS) generated by mitochondrial respiration constitute a major potential cause of DNA damage; low levels of ROS are required to maintain stem cells, while higher levels lead to differentiation. Researchers have observed a high capacity for antioxidant defense in murine stem cells, as well as greater efficiency of DNA repair. In their current report, Saretzki and colleagues examine changes in DNA maintenance and ROS production in differentiating human embryonic stem cells (hESCs). Using three fluorescent probes—dichlorodihydrofluorescein (DCF, a nonspecific probe for oxidative stress), dihydrorhodamine 123 (DHR, which is oxidized by peroxides and peroxynitrite), and the MitoSOX™ Red reagent (to assess superoxide production)—the authors observed an increase in ROS production during hESC differentiation. The authors also observed a loss of telomere maintenance and reduced DNA damage repair during differentiation; though no causal relationship among these three factors was established, the authors suggest these effects constitute closely related responses to a common set of differentiative cellular stimuli.
View bibliography reference
To study the mutualism between vertebrates and their associated microbial communities, or microbiota, the Guillemin lab has developed a germ-free zebrafish model. Our analysis has revealed important roles for the gut microbiota in intestinal epithelial maturation, cell homeostasis and cell type specification, and the establishment of mucosal tolerance. We are currently investigating the molecular nature of the microbiota-derived signals that drive these developmental programs. One interesting phenotype we are exploring is the reduced rate of intestinal cell proliferation in zebrafish larvae reared without microbes. Typically, BrdU labeling is used to determine the number of proliferating cells in situ, but it is often not reliable and the harsh tissue treatment affects labeling with other antibodies.
In this image, a 5-day-old zebrafish larva was reared in a germ-free environment and then exposed to a 16 hour pulse of 400 μM EdU, a nucleoside analog which is incorporated into cells that are actively synthesizing DNA. The tissue was paraformaldehyde fixed for 4 hours at room temperature then immediately paraffin embedded, and cut in 7 micron sections. After deparaffinization, rehydration, permeabilization, and a 10 minute blocking step in 3% BSA + 0.5% Triton® X-100 in PBS, the tissue was labeled by the click reaction cocktail. The incorporated EdU was then labeled with the azide-modified click detection reagent for 30 minutes. Slides were then rinsed, blocked with 1%BSA/ 0.3% Triton® X-100 for an additional 15 minutes prior to staining with 20 mg/mL lectin soybean agglutinin (SBA) Alexa Fluor® 568 conjugate in blocking buffer.
The image is a cross section through the mid intestine. Proliferating nuclei appear white due to labeling with both the green Alexa Fluor® 488 azide from the Click-iT™ EdU Alexa Fluor® 488 Imaging Kit and blue from the nuclear counterstain TO-PRO®-3 dye which detects all nuclei in the tissue. Goblet cells in the intestinal bulb are labeled with lectin soybean agglutinin Alexa Fluor® 568 conjugate (red), which stains the mucus compartment. The large circular void in the lower center of the image is the intestinal lumen; numerous actively proliferating intestinal cells can be seen in the surrounding epithelial wall in white. The 20× z-section image was captured using a CoolSNAP camera (Photometrics Systems) mounted on a Nikon D-Eclipse C1 confocal microscope (2 msec (micro second) exposure time). The image was processed (including pseudocoloring) using Photoshop® software (Adobe, Inc.). Image submitted by Sarah Cheesman, Institute of Molecular Biology, University of Oregon, USA.
|Alexa Fluor® dyes page
Finding Alexa Fluor® conjugates just got easy with the Alexa Fluor® dyes page.
There, you'll be able to read why Alexa Fluor® dyes are the best for antibodies and other conjugates and quickly find Alexa Fluor® conjugates by research area application or product type.
Also, you’ll find quick links to the Molecular Probes® resources you have used in the past—Spectra Viewer, fluorescence and flow cytometry tutorials, and the Image Gallery. Explore the Alexa Fluor® dyes page.
PathwaysOnline is a new monthly electronic newsletter from Invitrogen. This publication focuses on specific cellular pathways and provides you with information on the latest products and innovative technologies so that you can make the most of every cell. Stay on the cutting edge of your pathway-based research. Subscribe to PathwaysOnline.
Biofilm science is a hot topic, and research into the identification, behavior, and management of biofilms is moving rapidly. Many of our Molecular Probes® fluorescence-based dyes and probes have proven very useful for imaging biofilms. In recent results, scientists at the Center for Biofilm Engineering used one of our stains to discern whether a large molecule antibiotic, daptomycin, could penetrate thick methicillin-resistant Staphylococcal biofilms (click the “Poster Spotlight” link in the upper right).