Here's what's in this issue:

Cellular Lights™ reagents for monitoring drug-induced cytoskeletal changes in living primary cells

what they are
Cellular Lights™ reagents are ready-to-use solutions of baculovirus that utilize the BacMam technology to deliver a particular fluorescent protein to a specific subcellular location in mammalian cells. These reagents are useful in functional cell biology imaging applications to easily highlight subcellular structures with imaging or HCS instruments.

how they work
Normal human aortic smooth muscle cells (Cascade Biologics, C-007-5C ) were transduced with Cellular Lights™ Tubulin-GFP (green) according to the new “add and go” protocol for easily transduced cell types. By using this protocol, the GFP-tubulin construct is  efficiently and reproducibly delivered to a range of cell types. Twenty-four hours after transduction, cells were stained with the nuclear dye, Hoechst 34552 (blue), and treated with 20 µM vinblastine, a well-known antimitotic drug used in the treatment of cancer. Before the addition of vinblastine, tubulin adopts a canonical "long fiber" form. However, 30 minutes after vinblastine addition, microtubule assembly is inhibited and small structures resembling grains of rice are formed (see figures).

Normal human aortic smooth muscle cells transduced with Cellular Lights™ Tubulin-GFP and treated with vinblastine. The image on the left was taken at the time of treatment; the image on the right was taken 30 minutes after treatment.

what they offer

  • ability to view subcellular structures in living cells
  • simple transduction protocol
  • compatibility with both imaging and high-throughput imaging (HCS) platforms


Visit us online to find out more about Cellular Lights™ reagents.

PerCP conjugates for flow cytometry

what they are
Primary antibodies conjugated to PerCP with validation for flow cytometry.

how they work
Peridinin chlorophyll protein (PerCP) is a fluorochrome popularly used as third color on 488 nm laser–equipped flow cytometers. PerCP is excited by the 488 nm laser, with peak emission at 678 nm, corresponding to FL3 on BD FACScan, FACSCalibur, and FACSCanto cytometers, and FL4 on Beckman Coulter instruments. Invitrogen now offers direct conjugates of PerCP primary antibodies for flow cytometry applications.

what they offer
  • A third color option for single laser cytometers
  • Excited by the 488 nm laser, and detected in the red channel (FL3 on most instruments)
  • Matching isotype controls

To browse antibodies by specificity or application visit www.invitrogen.com/antibodies. Find antibodies and other reagents for flow cytometry at www.invitrogen.com/flowcytometry.

Product Regulatory status Quantity Cat. no.
Mouse anti-human CD45 PerCPASR0.5 ml MHCD4531
Mouse anti-human CD3 PerCPASR0.5 ml MHCD0331
Mouse anti-human CD19 PerCPASR0.5 ml MHCD1931
Mouse IgG1 isotype controlASR0.5 ml MG131
Mouse IgG2a isotype controlASR0.5 ml MG2a31


Human peripheral blood lymphocytes were stained with PerCP conjugated anti-human CD45 monoclonal antibody. The negative profile represents unstained cells.



Human peripheral blood lymphocytes were stained with PerCP conjugated anti-human CD19 monoclonal antibody. The negative profile represents unstained cells.



Human peripheral blood lymphocytes were stained with PerCP conjugated anti-human CD3 monoclonal antibody. The negative profile represents unstained cells.




Uncover the phenomenon of insulin signaling



what they are

The molecular signaling mechanisms by which insulin leads to increased glucose transport and metabolism and gene expression are not completely elucidated. Invitrogen immunoassays can help you uncover the mysteries of insulin signaling. Perform quantitative analysis of phosphoproteins in the insulin signaling pathway using our ELISA kits or, for simple and efficient profiling, use the Luminex® Multiplex assays that require only 10,000 cells.

how they work
Insulin possesses a wide spectrum of biological actions, it stimulates cellular glucose uptake, glucose oxidation, glycogenesis, lipogenesis, proteogenesis and the formation of DNA and RNA. Insulin receptor substrate 1 (IRS 1), plays a key role in mediating metabolic and proliferative signaling arising from stimulation by insulin, IGF 1, IGF 2, and cytokines such as IL 4.


For accurate quantitation of intracellular or extracellular proteins, Invitrogen offers a wide range of immunoassays, antibody pairs, and ELISA kits that provide single-protein measurements, while the Multiplex Luminex® assay is designed to simultaneously quantify the levels of multiple proteins. Both ELISA and Multiplex Luminex® assays are solid-phase sandwich immunoassays for measuring phosphorylated/total proteins in cellular extracts. In the ELISA kits, the solid support is the microtiter plate (precoated with monoclonal antibodies), whereas in the Luminex® Multiplex assays beads of defined spectral properties (conjugated to analyte-specific capture antibodies) serve as the solid support. For the Luminex® Multiplex assay, the conjugated beads and samples are added to the wells of a filter-bottom microplate and are incubated. During this first incubation, the target analyte (or analytes) is captured. After washing, a biotinylated anti-rabbit secondary antibody (with specificity to the target analyte) is added. After the secondary antibody is allowed to bind, detection is achieved by the addition of streptavidin R-PE (R-phycoerythrin). After washing to remove unbound anti–rabbit R-PE, the beads are analyzed with the Luminex® 100™ or 200™ instrument.

what they offer
  • superior performance—accurate, reproducible, and sensitive quantitation of insulin signal transduction 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

Visit us online to find our complete list of immunoassays.


Antibody pair
Product Quantity Cat. no.
Glycogen Synthase [pSpS641/645] Antibody Pair5 plates CHO0225
Glycogen Synthase-1 (Total) Antibody Pair5 plates CHO0265
IRS-1 (Total) Antibody Pair5 plates CHO0195

ELISA
Product Quantity Cat. no.
Glycogen Synthase-1 (Total) ELISA Kit96 tests KHO0721
Glycogen Synthase [pSpS641/645] ELISA Kit96 tests KHO0731
GSK-3β [pS9] ELISA Kit96 tests KHO0461
GSK-3β (Total) ELISA Kit96 tests KHO0451
IGF-1R (Total) ELISA Kit96 tests KHO0491
IGF-1R [pYpY1135/1136] ELISA Kit96 tests KHO0501
Insulin Human ELISA Kit96 tests KAQ1251
IR Human (Total) ELISA Kit96 tests KHR9111
IR Human [pY1158] ELISA Kit96 tests KHR9121
IR Human [pY1328] ELISA Kit96 tests KHR9151
IR Human [pY1334] ELISA Kit96 tests KHR9161
IR Human [pY972] ELISA Kit96 tests KHR9141
IR Human [pYpY1162/1163] ELISA Kit96 tests KHR9131
IRS-1 (Total) ELISA Kit96 tests KHO0511
IRS-1 [PS312] ELISA Kit96 tests KHO0521
IRS-1 PY612 ELISA Kit96 tests KHO0931

Luminex® assays
Product Quantity Cat. no.
Glycogen Synthase-1 (Total) Singleplex Bead Kit100 tests LHO0721
Glycogen Synthase-1 [pSpS641/645] Singleplex Bead Kit100 tests LHO0731
GSK-3β [pS9] Singleplex Bead Kit100 tests LHO0461
GSK-3β (Total) Singleplex Bead Kit100 tests LHO0451
IGF-1R (Total) Singleplex Bead Kit100 tests LHO0491
IGF-1R [pYpY1135/1136] Singleplex Bead Kit100 tests LHO0501
Insulin Human Singleplex Bead Kit100 tests LHP0021
IR (Total) Singleplex Bead Kit100 tests LHR9111
IR [pYpY1162/1163] Singleplex Bead Kit100 tests LHR9131
IRS-1 (Total) Singleplex Bead Kit100 tests LHO0511
IRS-1 [pS312] Singleplex Bead Kit100 tests LHO0521

Primary antibodies against mitochondrial Complex I subunits

what they are
Invitrogen offers a number of specific primary antibodies against mitochondrial proteins, including Complex I subunits.

how they work
The study of mitochondrial Complex I proteins (also known as NADH ubiquinone oxidoreductase) is important not only for basic cellular research, but also for clinical diagnoses. Research in this area has taken on greater significance since it has been discovered that many diseases originate from dysfunctional mitochondrial Complex I proteins. Several neurological disorders including Parkinson’s disease and schizophrenia as well as inherited genetic disorders can be traced back to functional or structural defects of Complex I. The proteins of Complex I also show sensitivity to many pesticides such as acaricides and rotenone. Complex I comprise of 45 to 46 different subunits, and Invitrogen currently offers antibodies for several of these (and a continually increasing portfolio of new ones).

what they offer

  • superior specificity for mitochondrial proteins.
  • validated reagents with multiple applications and species specificities
  • part of a wide-ranging portfolio of antibodies for mitochondrial research.

For a complete list of mitochondrial research antibodies visit www.invitrogen.com/antibodies.


Figure A



Figure B
A:  Figure shows the Complex I GRIM19 subunit location in HDFn cells. Complex I GRIM19 subunit is stained red with the help of Anti-Complex I GRIM-19 Monoclonal Antibody (Cat. no. 438900) followed by an Alexa Fluor® 594 dye-conjugated goat anti–mouse IgG secondary antibody. Nuclei are shown in blue.

B: 
Western blot showing GRIM 19 Complex I subunit band at ~19 kDa, detected using anti-Complex I GRIM-19 Monoclonal Antibody (Cat. no. 438900) followed by an HRP-conjugated goat anti–mouse IgG secondary antibody.

Product Reactive species Application Quantity Cat. no.
Anti-OXPHOS Complex I 30Hu, B, Ms WB100 μg 459130
Anti-Complex I NDUFA9 Mono AbHu, Ms, B, Rt WB, IF/ICC 100 μg459100
Anti-Complex I Monoclonal AbHu, Mo, Ch, B IP100 μg438800
Anti-Complex I GRIM-19 Mono AbHu, B, Rt WB, ICC, IHC 100 μg438900
Anti-Complex I NDUFS4 Mono AbHu, Ms, B, Rt WB100 μg439000
Anti-Complex I NDUFS3 Mono AbHu, B, Ms, Rt WB100 μg439200
Anti-Complex I Mitoprofile AbHuIP100 μg456925

SYTO® dyes find multiple uses in flow cytometry

Invitrogen’s Molecular Probes® SYTO® stains are widely used in the research community with countless citations to their credit. The June 2008 issue of Cytometry (Vol 73A), Part A features three articles highlighting just a few of the applications for Molecular Probes® SYTO® dyes. The following is a summary of the findings presented.
 
SYTO Dyes and Histoproteins-Myriad of Applications, Tarnok, A. (2008) Cytometry 73A:477-479.
The editor of Cytometry, Attila Tarnok, writes about the growth of applications for the SYTO® family of dyes in the June issue of Cytometry. Starting with a review of the variations in the chemical structure that explain the unique behaviors of some of the members of the SYTO® dye family, Tarnok reviews the widely published utility of the popular SYTO® 13, SYTO® 16, and SYTO® 14 green light–excitable stains. He continues by saying that the use of the SYTO® green-fluorescent stains in flow cytometry is well established, but that orange-excitable, red-excitable, and blue-excitable SYTO® stains are gaining recognition with the increasing use of varied excitation lasers. The value of Wlodkowic’s work (see below) with the orange light– and red light–excitable SYTO® stains is also reviewed. In particular, he notes the use of SYTO® stains for multiparameter applications in flow cytometry and the potential for cytometric and microfluidic HCA applications.

SYTO Probes in the Cytometry of Tumor Cell Death, Wlodkowic, D et.al. (2008) Cytometry 73A:496-507.
Wlodkowic et al., provide the first characterization of the orange light–excitable SYTO® dyes (80-81) for flow applications. In this paper Wlodkowic and colleagues report the use of these SYTO® stains in combination with a necrotic cell marker to discriminate viable, apoptotic, and necrotic cells. The authors note the value of high-throughput assays that permit quantitative multivariate characterization of cellular apoptotic events. 

Multiparameter Detection of Apoptosis Using Red-Excitable SYTO Probes, Wlodkowic, D et.al. (2008) Cytometry 73A:477-479.
The utility of the red light–excitable SYTO® dyes (17, 59–64 ) for multiparameter analysis of the apoptotic process is reviewed for the first time by Wlodkowic et al. This manuscript reviews the staining characteristics and highlights the advantages of SYTO® stains, which include reduced need for spectral compensation, low cost, and simplicity of the staining protocols. These attributes make these SYTO® stains particularly useful for flow cytometric applications. Notably, the authors demonstrate the utility of SYTO® 62 for the specific detection of apoptotic stimuli.

The mitotic kinesin CENP-E is a processive transport motor.
Yardimci, H. et al. (2008) Proc Natl Acad Sci 105: 6016–6021.

What sort of motor proteins govern the movement of chromosomes during mitosis? Supplied with sufficient ATP, the motor protein kinesin-1 can take more than a hundred ~8 nm steps along a microtubule without dissociating, generating forces of 5–7 pN as it travels. This type of movement, known as processive motility, plays a role in the long-distance transport of vesicles and organelles. Centromeric protein E (CENP-E)—a kinetochore-associated member of the kinesin superfamily—is necessary for capturing and positioning chromosomes to the mitotic spindle during metaphase. While many mitotic kinesins are characterized by motion that is either minimally or non-processive, CENP-E shares structural traits both with processive and non-processive kinesins, making it difficult to define how this protein functions in vivo. In their recent study, Yardimci and colleagues used Qdot® 655–labeled CENP-E dimers to enable the observation of CENP-E motility along axonemes by total internal reflection fluorescence (TIRF) microscopy. They reported an average run length for labeled CENP-E that is comparable to the distance of observed metaphase chromosomal movement, noting that most CENP-E motors reached the end of the axoneme structure before dissociating. The authors also reported other similarities between CENP-E and kinesin 1, namely, nearly identical kinetic parameters for ATP utilization and comparable step size, motion, and force (8.4 nm steps in a hand-over-hand fashion, generating ~6 pN of force). The authors see these biophysical measurements as strong support for previous observations regarding the mechanism of chromosomal movement during mitosis.

View bibliography reference

 


Flagellophora cf. apelti were exposed to the nucleoside analog EdU (5-ethynyl-2'-deoxyuridine) for 10 hours (100 µM in sea water). Following fixation and permeabilization, EdU that had been incorporated into newly synthesized DNA was detected with the Click-iT® EdU Alexa Fluor® 488 Imaging Kit (A10083, green fluorescence). Phospho-H3 was detected using a rabbit primary antibody followed by an Alexa Fluor® 568 dye–labeled goat anti–rabbit IgG antibody (A11011, red fluorescence). Dr. Smith's lab studies stem-cell dynamics in primitive flatworms; Flagellophora belongs to the group Nemertodermatida (arguably the most primitive group of flatworms) whose stem cells (neoblasts) are woefully understudied. As the animals are difficult to collect and do not survive long after extraction from the sand, the simplicity of S-phase stem-cell labeling with the Click-iT® EdU Kit has proven very helpful in this research. In addition, the ability to carry out subsequent antibody labeling (in this case, anti–phospho-H3 for M-phase cells) is a big improvement over traditional methods of S-phase labeling that destroy antigenicity. The image was submitted by Julian Smith, Department of Biology, Winthrop University, USA.

 

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

Learn more about the TLR Pathway.



 Find the right antibody with our newly designed antibodies search page
Looking for antibodies? Check out the newly designed Antibodies and Secondary Detection web page. You can search by gene, or browse by cell process, research area, dye type, antibody type, and more. Using this search, you can also quickly find secondary antibodies. Search for antibodies.


 Introducing BioPath Online
BioPath Online 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 BioPath Online.

 


 More and more researchers rely on the Qubit® fluorometer for accurate quantitation
The Qubit® fluorometer combined with Quant-iT™ reagent kits form the Qubit™ Quantitation Platform—a consistent, simple, and accurate quantitation system for DNA, RNA, and protein samples. But don’t take our word for it. Here’s what one of our customers has to say about the Qubit™ Quantitation Platform:

“For production we need to make a large mass of DNA, and we need to measure the concentration accurately before aliquoting. Our production methodology yields large amounts of RNA along with the DNA. We treat the sample with RNaseA, but even after treatment the individual RNA monomers still absorb at 260 nm, so we can’t use a spectrophotometer to quantify the DNA. This is where the Qubit comes in. We can use it to accurately quantify the DNA in the presence of the digested RNA since the dye only binds to DNA. We looked at the measurement relative to the intensity of known standards on a gel and the Qubit appears to be quite accurate.

Mark G. Wise, Ph.D.
Staff Scientist Bacterial Barcodes, Inc. (a wholly-owned subsidiary of BioMerieux, Inc.)”

You, too, can get the most accurate quantitation possible, starting today! Find out more about the Qubit® fluorometer.

 



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