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Rhod-3 AM Imaging Kit

what it is
Rhod-3 AM dye (supplied in the Rhod-3 AM Imaging Kit) offers significant improvements over existing Ca2+-sensing dyes. Its uptake into organelles is reduced, which translates to better cytosolic localization. Also Rhod-3 AM exhibits a large increase in fluorescence emission upon binding to calcium, making it more sensitive than other red-fluorescent calcium dyes. The novel Rhod-3 AM Imaging Kit is designed for live-cell imaging of cytosolic calcium signaling in combination with green fluorescent dyes or proteins. Red-shifted calcium dyes are also applicable where CFP/YFP are used as FRET-based biosensors.

how it works
The dye in the Rhod-3 AM Imaging Kit is a rhodamine derivative attached to a calcium chelator, and is supplied as an AM ester to facilitate entry into cells. Once inside the cell, intracellular esterases cleave the AM group, trapping the dye in the cell and rendering the dye fluorescent. As calcium binds, the fluorescence intensity of the dye is enhanced. The spectral properties of Rhod-3 AM (ex/em = ~560/600 nm) and strong fluorescence upon calcium binding gives an excellent signal window and makes it our brightest red calcium dye.

what it offers
  • narrow emission enables simultaneous detection with GFP or other dyes
  • compared to existing red calcium dyes, Rhod-3 AM exhibits a significantly higher increase in fluorescence upon calcium binding
  • enhanced cytosolic localization for better detection of cytosolic transients



Excitation and emission peaks of Rhod-3 AM (panel A). The new red-shifted calcium dye, Rhod-3 AM, is the preferred choice for multiplexing with GFP (panels BI and BII). Rhod-3 AM can also be imaged simultaneously with other green fluorophores. Panel C shows HeLa cells that have been co-loaded with Rhod-3 AM (10 µM) and mag-fluo-4 AM (5 µM) for simultaneous monitoring of cytosolic and intra-ER calcium (for detailed methods see Park et al. 2000 EMBO J 19:5729–5739).

ELISA Kits for p27 and p21 proteins 

what they are 
Invitrogen is now offering two new assay kits to measure p27 Kip1 and p21 Waf1/Cip1 protein levels in cell lysates. As cyclin-dependent kinase (CDK) inhibitors, both proteins are negative cell cycle regulators that play an important role in tumor suppression. Cell division relies on the activation of cyclins, which bind to CDKs to induce cell cycle progression towards S phase and later to initiate mitosis. Uncontrolled CDK activity is often the cause of human cancer; CDK function is tightly regulated by cell cycle inhibitors such as p21 Waf1/Cip1 and p27 Kip1 proteins. Following anti-mitogenic signals or DNA damage, p21 Waf1/Cip1 and p27 Kip1 bind to cyclin–CDK complexes to inhibit their catalytic activity and induce cell cycle arrest. 

how they work 
The ELISA kit method offers a simple and unbiased way to quantify specific proteins. The Invitrogen ELISA kit employs a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA); antibodies are coated to the bottom of a 96-well plate (immobilized); the sample is added to the well, and the immobilized antibody binds to the protein antigen. Nonspecifically bound proteins are washed away. A detector antibody is then added, followed by a secondary antibody conjugated to horseradish peroxidase (HRP). Finally a chromogenic substrate is added that reacts with HRP to produce color, the intensity of which is directly proportional to the concentration of the protein present. A stop solution is added to terminate the color reaction, and the intensity is measured using a plate reader. Invitrogen ELISA kits come ready-to-use with all the necessary reagents including calibrated standards. 

what they offer

  • sensitive—use a small sample volume
  • specific—measure the protein you're interested in
  • fast—4 hours to complete
  • minimal variation—consistent lot-to-lot performance


 Figure 1—Natural p27 Kip1 from serum-starved MCF-7 cell lysate was serially diluted in Standard Diluent Buffer (provided with the kit). The parallelism demonstrated between the standard and MCF-7 lysate indicates that the standard accurately reflects p27 Kip1 content in samples.

 Figure 2—Apicidin treatment (2 µg/ml for 24 hours) increases p21 Waf1/Cip1 expression level in MCF-7 cells. Untreated MCF-7 cells were used as control. Cell extracts were prepared and cell lysates (200 mg/ml) were analyzed with p21 Waf1/Cip1 ELISA Kit (Cat. no. KHO5421).

Product Name Cat. No.
p21 Waf1/Cip1 ELISA Kit, 96 testsKHO5421
p27 Kip1 ELISA Kit, 96 testsKHO5321

Learn more about our ELISA kits.

Akt Pathway Phospho 7-Plex Panel and the Luminex® 200™ detection system

what it is

The insulin receptor IR/IGF-1R–Akt signaling pathway is crucial for regulating cell survival, proliferation, and a number of insulin metabolic effects. Perturbations in this pathway are suspected as key indicators (and perhaps root causes) of diseases such as cancer and diabetes. A method to simultaneously measure the activity of these proteins (IR [pYpY1162/1163], IGF-1R [pYpY1135/1136], IRS-1 [pS312], Akt [pS473], GSK-3β [pS9], PRAS40 [pT246], p70S6K [pTpS421/424]) will significantly expedite drug discovery efforts that target this pathway. Invitrogen has developed the Akt Phospho 7-Plex Panel (LHO0001) and now offers the Luminex® 200™ detection system with xPONENT® software to meet this need.

how it works

The Akt Phospho 7-Plex Panel is a multiplex bead immunoassay designed to determine the phosphorylation state of seven proteins in the IR/IGF-1R–Akt signaling pathway: IR [pYpY1162/1163], IGF-1R [pYpY1135/1136], IRS-1 [pS312], Akt [pS473], GSK-3β [pS9], PRAS40 [pT246], and p70S6K [pTpS421/424]. The assay is a solid-phase sandwich immunoassay that is designed to be analyzed using the Luminex® 100™ or 200™ instrument. The spectral properties of 100 distinct bead regions can be monitored with the Luminex® 100™ or 200™ instrument, a capability that affords this assay system the potential for measuring up to 100 different analytes in a single sample. Beads of defined spectral properties conjugated to analyte-specific capture antibodies and samples (including standards of known analyte concentration, control specimens, and unknowns) along with Detector Antibody are pipetted into the wells of a filter-bottom microplate and incubated for 3 hours. During this first incubation, analytes bind simultaneously to the immobilized (capture) antibodies and to the solution-phase rabbit polyclonal (detector) antibodies. After washing, an anti–rabbit R Phycoerythrin (R-PE)– conjugated secondary antibody is added for 30 minutes. During this final incubation, the anti–rabbit R-PE binds to the detector antibodies associated with the immune complexes on the beads, forming four-member, solid-phase sandwiches. After washing to remove unbound anti–rabbit R-PE, the beads are analyzed with the Luminex® 100™ or 200™ instrument. By monitoring the spectral properties of the beads and the amount of associated R-PE fluorescence, the concentration of the seven analytes can be determined.


what it offers

  • superior performance—accurate, reproducible, and sensitive quantitation of the Akt pathway proteins
  • high quality—in-house manufactured antibodies ensure excellent specificity and sensitivity
  • fast and easy protocols—perform and analyze your data in less than one day

 

  Product name Cat. no.
 Akt, JNK1/2, p38 MAPK Phospho 3-Plex Panel, 100 testsLHO0061
new and
improved
Akt Pathway Phospho 7-Plex Panel, 100 testsLHO0001
 Akt, JNK1/2, p38 MAPK Total 3-Plex Panel, 100 testsLHO0051
 Akt Pathway Total 7-Plex Panel, 100 testsLHO0002
newLuminex® 200 with xPONENT® software, one systemMAP0200


Learn more about Luminex® immunoassays. To learn more about the Luminex® 200™ detection system, visit www.invitrogen.com/luminexinstrument.

Phosphorylation site–specific antibodies for platelet-derived growth factor receptor (PDGFR)

what they are
Invitrogen offers monospecific reagents for studying the phosphorylation of platelet-derived growth factor receptor (PDGFR), and new additions cover both alpha and beta isoforms.

how they work
Phosphorylation is a common mechanism for activation and deactivation of proteins in cell signaling cascades. Phosphorylation site–specific antibodies (PSSA) that are monospecific for the phosphorylation state of a particular tyrosine, serine, or threonine residue on the target protein are critical for elucidating these signaling pathways. Invitrogen offers antibodies against all isoforms of PDGFR to monitor protein expression and regulatory phosphorylation of primary activation sites.

 Up-regulation and antibody-peptide competition
Extracts of NIH3T3 cells unstimulated (1) and stimulated with 50 ng/ml PDGF for 10 minutes (2–5) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4°C, then incubated with the PDGFRα [pY754] antibody for two hours at room temperature in a 1% BSA-TBST buffer, following prior incubation with: no peptide (1, 2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphotyrosine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F(ab´)2 anti–rabbit IgG alkaline phosphatase (Cat. no. ALI4405) and signals were detected using the Tropix WesternStar™ method. The data show that only the phosphopeptide corresponding to PDGFRα [pY754] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of PDGFRα [pY754] phosphorylation by the addition of PDGF in this cell system.


what it offers

  • validated reagents with multiple applications and species specificities
  • wide range of targets and modification sites
  • all phosphorylation site-specificities are verified with peptide competition

 

Product name Size Species Applications Cat. no.
PDGFR α [pY754] PAb10 blotMs (Hu, Rt)WB44-1008G
PDGFR α [pY762] PAb ms10 blotMs (Rt)WB44-1010G
PDGFR β [pY751] PAb10 blotHu, Ms (Rt)WB44-1013G  
PDGFR α [pY572/pY574] /
β [pY579/pY581] PAb
10 blotMs, Hu, RtWB44-1000G  
PDGFR β [pY1009] PAb10 blotHu, Ms (Rt)WB44-1016G  
PDGFR β [pY771] PAb 10 blotHu, Ms (Rt)WB44-1014G  
PDGFR β (pY716) PAb hu, ms (rt)10 blotHu, Ms (Rt)WB44-1011G
PDGFR β [pY740] PAb10 blotHu, Ms (Rt)WB44-1012G  

PDGFR α [pY572/pY574] /
β [pY579/pY581]

10 blotMs, Hu, RtWB44-1000  
PDGFR α [pY742] PAb10 blotMs (Hu, Rt) 44-1006
PDGFR α [pY754] PAb10 blotMs (Hu, Rt)WB44-1008
PDGF α [pY849] / β [pY857]10 blotHu, MS, Rt, CkWB44-1001G
PDGF-D Rb100 μgHu, Rt, MsWB40-2100


Learn more about phosphorylation site–specific antibodies. Browse antibodies by specificity or application visit www.invitrogen.com/antibodies.

Primary antibodies against mitochondrial Complex II subunits

what they are
Invitrogen offers wide variety of tools for mitochondrial research. These include specific primary antibodies against mitochondrial proteins that allow the researchers to study mitochondrial proteins, including Complex II subunits.

how they work
Invitrogen now offers the most complete portfolio of antibodies for mitochondrial research, and continues to add new antibodies, kits, and controls for the study of mitochondrial oxidative phosporylation (OxPhos), electron transport chain, and Krebs cycle among other mitochondrial pathways. Mitochondrial Complex II (succinate:ubiquinone oxidoreductase) is the smallest complex in the respiratory chain and comprises four subunits. Invitrogen offers antibodies against most of the OxPhos proteins and also against other mitochondrial proteins.

what they offer
  • best specificity for mitochondrial proteins
  • validated reagents with multiple applications and species specificities
  • part of a wide-ranging portfolio of antibodies for mitochondrial research



Localization of Complex II 70 kDa Fp subunit in the HDFn cells.  Complex II 70 kDa Fp subunit is shown in green color with the help of Anti-Complex II 70 kDa Fp subunit Monoclonal Antibody (Catalog # 459200) and Alexa 488-conjugated goat anti-mouse secondary antibody. Nucleus is shown in Blue.

Product name Reactive species Application Cat. no.
Complex II 70 kDa Fp subunit Monoclonal Antibody, 100 μgMs, Hu, B, Rt, CeWB, IF/ICC459200
Complex II Monoclonal Antibody, 100 μgHu, B, MsIP439300
Complex II Mitoprofile® Antibody, 100 μgHu, B, Ms, RtIP457025
Hu Complex II Mitoprofile® Antibody Cocktail, 100 μgHu, B, Ms, RtWB457830
OxPhos Complex II 30 kDa subunit, 100 μgHu, B, Ms, RtWB, IFA21345

To see all of the antibodies Invitrogen offers for mitochondrial research visit  www.invitrogen.com/antibodies.

Endothelial cilia are fluid shear sensors that regulate calcium signaling and nitric oxide production through polycystin-1.
Nauli, S.M. et al. (2008) Circulation 117: 1161–1171.

How do vascular endothelial cells sense shear stress? Significant evidence exists to suggest that physiologic fluid shear stress offers beneficial effects in vivo. For example, vascular regions with steady laminar flow are generally protected from atherosclerosis, and shear stress has been shown to upregulate the expression of vascular endothelial growth factors in osteoblasts. Vascular endothelia are known to release nitric oxide—a potent vasodilator—in response to fluid shear, but the specific mechanosensory apparatus that mediates this response is unknown. In their present study, Nauli and colleagues investigated the role of primary cilia in the activation of shear stress–related physiological response in vascular endothelia. Immunofluorescence staining identified the proteins polycystin-1 and polaris—known components of primary cilia—in aorta of embryonic mice. Cells were observed to respond to applied shear stress by transiently releasing intracellular calcium and nitric oxide; mutant cells defective in cilia structure/function showed no response to shear stress. Prolonged shear stress led to polycystin-1 cleavage in wild-type cells, accompanied by the loss of the shear stress response. In an effort to further elucidate the roles of polycystin-1 and polaris in the shear stress response, cells were subjected to other physical/chemical stimuli, namely apical membrane distortion and exposure to acetylcholine. Both wild-type and mutant cell types showed similar responses to these stimuli despite their different shear stress responses, indicating a role for native polycystin-1 and polaris proteins that is specific to the shear stress signaling mechanism.

View bibliography reference 
HeLa cells were transduced with Organelle Lights™ Endosome-GFP (green fluorescence) and Organelle Lights™ Lysosome-RFP (red fluorescence). Cells were then loaded with 50 nM MitoTracker® Deep Red 633 (pink fluorescence). Imaging was performed using a Delta vision® Core microscope. Image was submitted by Nicholas Dolman, Invitrogen Corporation, Eugene, OR, USA.
New pathway web pages
Whether your IFN pathway research involves basic research tools, cell-based assays, or comprehensive screening services, Invitrogen has solutions for you. See our new IFN pathway web page for more information.


 New IFN pathway web page
Empower your research today using Invitrogen’s comprehensive portfolio of products and services to investigate the IFN pathway—everything from high-quality reagents for basic research and assay development to validated biochemical and cell-based assays, and world-class profiling and screening services.

See our portfolio of IFN pathway–associated reagents at www.invitrogen.com/IFN.






The Total Luminex® 200™ System

Invitrogen has pioneered one of the most comprehensive analyte panels in the industry, and now offers the complete solution by introducing the Luminex® 200™ detection system. Experience the power of Luminex® multiplex analysis all from Invitrogen. The Total Luminex® 200™ System is a compact analyzer that performs up to 100 assays simultaneously in a single well of a microtiter plate. This system is a flexible analyzer based on the principles of flow cytometry, which integrates key xMAP® detection components such as lasers, optics, advanced fluidics, and high-speed digital signal processors. Utilizing the xPONENT® software of the xMAP® technology operating system, the Luminex® 200™ System offers optional technical controls for 21 CFR Part 11 compliance to provide an electronic audit trail.

For more information, visit www.invitrogen.com/luminexinstrument


Validation of novel tools that detect the elusive O-GlcNAc

The O-GlcNAc (O-linked β-N-acetylglucoasamine) modification is an abundant, highly dynamic, intracellular regulatory modification found in all eukaryotic cells; like phosphorylation, O-GlcNAc modification plays a key role in signaling pathways. Until recently, the O-GlcNAc modification presented several formidable challenges for detection. First, O-GlcNAc is very small, uncharged molecule, so O-GlcNAc-modified proteins do not migrate any differently from unmodified ones. Additionally, being present at very low levels, it is often beyond the detection limits of antibody- and lectin-based detection methods. Finally, O-GlcNAc is very labile; even with mass spectrometry, the molecule simply goes undetected. Clark et al. recently reported on an advanced chemoenzymatic labeling strategy based upon click chemistry that overcomes the limitations of these existing techniques and enables detection of the dynamic O-GlcNAc at unprecedented levels (Clark PM et al., (2008) Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins. JACS (web release date August 7, 2008)). The authors initially tested the technique on α-crystallin (a known O-GlcNAc modified protein with a low extent (~10%) of glycoslyation) and detected femtomole levels with the new chemoselective approach (compared to conventional antibody and lectin techniques, by which only microgram levels were detectable). The new method was then applied to nuclear and cytosolic protein fractions from rat forebrain, and a total of 213 proteins, representing 67 known and 146 novel O-GlcNAc proteins, were identified. Besides detecting O-GlcNAc proteins directly in gels (which streamlines proteomic analyses), the authors also demonstrated the how this near-quantitative, simple, and robust two-step chemoenzymatic method can be applied to image O-GlcNAc proteins in cells and tissues.



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