- Read the latest issue of the ProbesOnline E-Newsletter.
what they are
Invitrogen offers a number of primary antibodies against cell junction proteins, including tricellulin.
how they work
Tight junctions form an important barrier of paracellular transport in epithelial cells, and the sealing of two adjacent cells at bicellular tight junctions is well described. The main structural components of bicellular tight junctions are claudins and occludins—tetra–membrane-spanning proteins. Less is known about the structure of tricellular tight junctions, a point where three adjacent cells are in contact with each other. Tricellulin is the first protein identified that specifically concentrates in tricellular tight junctions. Like claudins and occludins, tricellulin protein has four membrane-spanning domains, and it is highly expressed in epithelium-derived tissues such as small intestine, kidney, and lung.
- good specificity for imaging tricellular junctions
- validated reagents with multiple applications and species specificities
- part of a wide-ranging portfolio of antibodies for cell junction research
To browse antibodies by specificity or application, visit www.invitrogen.com/antibodies.
Figures show localization of tricellular junctions using an antibody specific to the carboxy-terminus of tricellulin, in green (Cat. no. 48-8400). Bi-cellular tight junctions are revealed by the localization of occludin, in red (Cat. no. 33-1500).
|Product||Quantity||Applications*||Species reactivity||Clonality||Cat. no.|
|Rabbit Anti-Tricellulin (C-term)||100 μg||WB, ICC||Hu, Ms, Rt, Cn, Ch, Bv, Eq, Mk||Polyclonal, ZMD.699 (Rabbit IgG)||488400|
|Rabbit Anti-Tricellulin (N-term)||100 μg||WB, ICC||Hu, Ms, Rt||Polyclonal, ZMD.698 (Rabbit IgG)||488300|
|Mouse Anti-Occludin||100 μg||WB, E, IF||Hu, Ms, Rt, Cn||Monoclonal, OC-3F10 (Mouse IgG1-κ)||331500|
|Mouse Anti-Occludin-FITC||100 μg||IF||Hu, Ms, Rt, Cn||Monoclonal, OC-3F10 (Mouse IgG1-κ)||331511|
|Mouse anti-Occludin-HRP||100 μg||WB, E||Hu, Ms, Rt, Cn||Monoclonal, OC-3F10 (Mouse IgG1-κ)||331520|
|Rabbit Anti-Occludin||100 μg||WB, E, IP, IF, IHC, ICC||Hu, Ms, Rt, Cn||Polyclonal, Z-T22 (Rabbit IgG)||711500|
|Mouse Anti-ZO-1||100 μg||WB, E, IF||Hu, Cn||Monoclonal, ZO1-1A12 (Mouse IgG1-κ)||339100|
|Mouse Anti-ZO-1-FITC||100 μg||WB, E, IF||Hu, Ms, Rt, Rb, Ch, Cn, X||Monoclonal, ZO1-1A12 (Mouse IgG1-κ)||339111|
|Rabbit Anti-ZO-1||100 μg||ICC||Hu, Ms, Rt, Cn, Gp||Polyclonal, Z-R1 (Rabbit IgG)||617300|
|Rabbit Anti-ZO-1 (Mid)||100 μg||WB, IF, IHC (frozen)||Hu, Ms, Rt, Cn||Polyclonal, ZMD.436 (Rabbit IgG)||402200|
|Rabbit Anti-ZO-1 (N-term)||100 μg||WB, IF, IHC||Hu, Ms, Rt, Cn||Polyclonal, ZMD.437 (Rabbit IgG)||402300|
|*WB = western blot, ICC = immunocytochemistry, IP = immunoprecipitation,
IHC = Immunohistochemistry, IF = immunofluorescence, E = ELISA.
what it is
The immune system is regulated and maintained through apoptosis, a normal process in which cell populations are deleted in response to self-recognition, failure to bind MHC, and cytokine/growth factor withdrawal. To facilitate detection of apoptosis, we have developed a multiplex sandwich immunoassay that permits the simultaneous detection of three important biomarkers: cytochrome c (a protein that normally resides within the intermitochondrial space that is released to the cytosol in response to apoptotic stimuli), cleaved caspase-3 [175/176] (an important reporter for initiator caspase activation), and cleaved poly (ADP-ribose) polymerase (PARP) [214/215] (an important reporter for caspase-3 activation).
how it works
The Invitrogen Apoptosis 3-plex Panel is a solid-phase sandwich immunoassay that is designed for use with the Luminex 100™ or 200™ instrument. Beads of defined spectral properties conjugated to analyte-specific capture antibodies, and samples (including standards of known analyte concentration, control specimens, and unknowns) are pipetted into the wells of a filter-bottom microplate and incubated for 2 hours. During this first incubation, analytes bind to the immobilized capture antibodies. After washing, a detector antibody is added to the mixture for 1 hour. At the end of this incubation, the beads are again washed and then incubated with an RPE conjugate (containing anti-rabbit RPE plus streptavidin RPE) for 30 minutes. During this final incubation, the RPE conjugate binds to the detector antibodies associated with the immune complexes on the beads, forming four-member solid-phase sandwiches. After washing to remove unbound RPE conjugate, the beads are analyzed with the Luminex 100™ or 200™ instrument. By monitoring the spectral properties of the beads and the amount of associated RPE fluorescence, the concentrations of the three analytes can be determined.
what it offers
- simultaneous quantitation of three key biomarkers of apoptosis
- microplate-based format for efficient sample processing
- Luminex platform analysis for sensitive, accurate analysis
Apoptosis 3-plex Panel ( LHO0007)
Assays for phospholipase A1 and A2 (PLA1 and PLA2)
what they are
As researchers examine the role of phospholipases in atherosclerosis and other diseases, Invitrogen’s fluorescence-based assays for PLA1 and PLA2 are finding a new application.
how they work
Blood plasma levels of cholesterol and their carrier particles, LDL and HDL, are critical in many disorders, from atherosclerosis and diabetes to Alzheimer’s and metabolic diseases. It has been observed that cholesterol-lowering drugs and dietary cholesterol control strategies have not eliminated hardening of the arteries or atherosclerosis. It is postulated that there are instead other more critical risk factors, especially for the 50 million people in the US alone suffering with metabolic disorders or obesity.
High on the list of risk factors are members of the A1 and A2 phospholipase families, including endothelial cell ligase (EL), a PLA1 found on endothelial cells lining arterial blood vessel walls, and secreted members of the phospholipase A2 family, which have been shown to play a significant role in modulating the levels and toxicity of plasma HDL and LDL, respectively. Up-regulation of EL (by cytokines and other proinflammatory drugs) lowers HDL levels while elevating free fatty acids and reactive cholesterol. However anti-inflammatory drugs can down-regulate this cell-surface enzyme and reverse this effect by elevating intact, healthy plasma HDL. In parallel, the proatherogenic role of PLA2 is postulated to occur by acting on oxidized apoB-LDL particles to form proinflammatory lysophosphatidylcholine and oxidized nonesterified fatty acids. Both of these roles are consistent with patchy foci of plaques on endothelial walls that are the hallmark of nascent disease.
Thus, lowered cholesterol may reduce some but not all of the negative consequences associated with their presence in blood. Instead, these proatherogenic enzymes are now considered prime and novel targets for controlling and monitoring the progression of many disease states, including coronary disease, which remains a serious health problem worldwide. Maintaining good HDL and reducing LDL isn’t the whole story: in the presence of agents that upregulate these enzymes, such as inflammation, additional bad outcomes are a risk.
Invitrogen offers Molecular Probes PLA1- and PLA2-selective substrates with good responsiveness (16-fold increase in fluorescence upon cleavage of the Red/Green PLA2-selective substrate, A10072) and greatly improved signal-to-noise ratios. These are true feed-and-read assays: simply add the substrates in liposomes to the microwell plate, wait 30 minutes, and read the increase in fluorescence (or decrease, for candidate inhibitor compounds). At pennies per well these assays are ideal for large screens, but sensitive and selective enough for downstream lead characterization. While the assays are excellent in microplate format, other studies have shown their potential utility in whole-cell and even whole-animal studies.
As the attention turns towards phospholipases and away from cholesterol-lowering regimes, Invitrogen’s PLA substrates offer a vital tool for your research. They are nonradioactive, antibody-free assays that don’t require mass spectrometry, just standard fluorescent microplate readers. We also offer kits and substrates for the other phospholipase family members, PLA-C and PLA-D. See the product list below for complete offerings and enzyme selectivity.
References: Curr Atheroscler Rep 6, 126 (2004); 126-30; Prostaglandins Leukot Essent Fatty Acids 72, 173 (2005); Curr Drug Targets 8, 1307 (2007); Circulation 117, 678 (2008); Circulation 116, 1832 (2007).
- PLA1 vs. PLA2 selectivity created by removal of the sn1 or sn2 cleavage sites
- fluorescence-based readouts for high sensitivity
- freedom from mass spectrometry and assays that require radioactivity and antibodies
- screens at pennies per well
phospholipase A1 and A2 (PLA1 and PLA2)
- Red/Green BODIPY PC-A2 (1-O-(6-BODIPY 558/568-aminohexyl)-2-BODIPY FL C5-sn-glycero-3-phosphocholine) *ratiometric phospholipase A2 substrate* (A10072)
- PED-A1 (N-((6-(2,4-DNP)amino)hexanoyl)-1-(BODIPY FL C5)-2-hexyl-sn-glycero-3-phosphoethanolamine) *phospholipase A1 selective substrate* (A10070)
- Amplex Red Phosphatidylcholine-Specific Phospholipase C Assay Kit - 500 assays (A12218)
- Amplex Red Phospholipase D Assay Kit - 500 assays (A12219)
Salic, A. and Mitchison, T.J. (2008) Proc Natl Acad Sci USA 105: 2415.
Can cell proliferation assays tell us more? Accurate assessment of the degree of DNA synthesis in proliferating cells provides important information in a wide range of pharmacological and regulatory studies. While the two methods currently used for measuring cell proliferation have given researchers useful insights, each method has its limitations. [ 3H]-thymidine labeling is laborious and slow, offers poor resolution, and suffers from all the potential health and waste-disposal concerns inherent in radioligand methodology. BrdU labeling is faster and more sensitive, and offers better resolution; however, the success of this approach necessitates extensive sample denaturation that can be difficult to reproduce and can significantly degrade sample structure. In the present study, Salic and Mitchison demonstrate the advantages of "click" chemistry—incorporation of 5-ethynyl-2'-deoxyuridine (EdU) followed by labeling with a fluorescent azide derivative—as an alternative to existing cell proliferation assays. Their data show that EdU is extensively incorporated into the DNA of proliferating NIH 3T3, HeLa, and Xenopus cells. Subsequent visualization of the fixed cells with Alexa Fluor 488 or Alexa Fluor 594 azide derivatives revealed efficient and highly reproducible labeling regardless of the Alexa Fluor dye used. Comparison with BrdU staining carried out on the same cells revealed excellent colocalization of the signal from the two methods; however, the click methodology yielded substantially more intense fluorescence, with excellent preservation of sample structure. The authors further demonstrate that the click methodology can be used to visualize proliferation in unfixed cells, large tissue/organ explants, and even in live animals, greatly expanding the scope and utility of this already promising approach.
|Empower your research today using Invitrogen’s comprehensive portfolio of products and services to investigate the Akt/mTor 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 Akt pathway–associated reagents at www.invitrogen.com/akt.
Learn more about the Akt Signaling Pathway.
The Qubit™ quantitation platform, a fluorescence-based method for measuring DNA, RNA, and protein, provides higher accuracy and higher sensitivity than UV absorbance–based quantitation does.
Why is this important for your research? Researchers who have converted to the Qubit™ quantitation platform have shared their thoughts below. You’ll see that this accuracy leads not only to better quantitation, but also to better experimental results downstream. The Qubit™ quantitation platform is used for microarrays, transfection, western blots, PCR, genotyping, and FISH as well as the following applications:
How the Qubit™ platform improved the workflow in your colleagues’ labs
- “Troubleshooting qPCR is easier when starting material is properly characterized, and results from different experimental conditions can be confidently compared knowing that the starting amount of template was exactly the same. Also helps first-strand cDNA reactions to perform optimally. The same general idea goes for DNA and protein as well.”
- “I’m able to measure things more quickly, so I measure more things or things at more steps.”
- “I have been able to really quantify my RNA and proteins. RNA was especially important because we are collecting from a very low cell number.”
- “I am more sure about the amount of sample I have.”
- “It’s made it easier to measure protein concentration.”
- “Better accuracy in quantitation of DNA or RNA yields more reproducible results.”
- “Using our UV spectrophotometer was a lot of extra work, so I dreaded measuring my samples. The Qubit is easy and accurate.”
- “Cloning speed and efficiency increased dramatically, and qRT-PCR is more precise.”
- “Yes, I can analyze my samples quickly and trust the results more completely.”
- “Faster quantification of DNA, faster sequencing.”
- “I no longer worry about losing sample to measuring how much there is.”
- “We are more confident in the accuracy of our quantitations. People perform quantitations more often rather than estimating.”
- “It has changed my workflow because it is more accurate and sensitive than UV spec. I am so happy with Qubit.”
- “I am more confident about my downstream design and results.”
- “We use it for routine PCR. It offers time savings and accuracy.”
Improve your workflow with the Qubit™ quantitation platform. Visit www.invitrogen.com/qubit today to learn more.