| Description | Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner.In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative).We offer recombinant Annexin V conjugated to a numerous fluorophores, as well as an Annexin V biotin conjugate which can be detected with fluorophore-labeled streptavidin. By binding to PS, fluorophores labeled Annexin V can be used to detect and quantify apoptotic cells via flow cytometry or fluorescence microscopy. The excitation and emission maxima of the Annexin V conjugates are summarized in the following table. Cat.No.Ex/Em (nm)Formatrp226056NABiotinrp225999401/422AF405rp226057490/525AF488rp226060650/668AF647rp226002681/704AF680rp226003752/776AF750rp226053498/517FITCrp226004410/455Pacific Bluerp226006647/665Cy5rp226054650/660APCrp226055565/575PErp226058565/670PE-Cy5rp226059565/774PE-Cy7Precautions1. Please try to avoid light when using to slow down the quenching of fluorescence.2. Propidium Iodide Solution is toxigenic and mutagenic; handle with care.3. Due to the calcium dependence of the Annexin V:PS interaction, it is critical to avoid buffers containing EDTA or other calcium chelators during Annexin V experiments.Instruction for use1. Dilute 10X Binding Buffer (A1372288) to 1X using distilled water (1 mL 10X Binding Buffer + 9 mL ddH2O).2. Wash cells twice with cold PBS and then resuspend the desired amounts of cells in Annexin V Binding Buffer at a concentration of 1.0-5.0 x 106 cells/mL.3. Add 5 µL of Annexin V-APC to 100 µL of the cell suspension. Stain with a viability dye, such as PI (P1373641; P1372285), 7-AAD (A1372406), or DAPI (D1372407) dyes, if desired.4. Gently vortex the cells and incubate for 10 min at RT (25°C) in the dark.5. Add 100 µL of 1X Binding Buffer to each assay. Analyze by flow cytometry within 1 hr... Read More | Inquire | Protein Purity≥85% by SDS PAGEExtinction CoeffA280 nm = 0.631 at 1.0 mg/ml for pure C1qMolecular Weight400,000 Da (18 chains)General DescriptionRat C1q is purified from pooled normal rat serum. C1q is part of the C1 complex, which is the first complement component in the classical pathway of Protein Purity≥85% by SDS PAGEExtinction CoeffA280 nm = 0.631 at 1.0 mg/ml for pure C1qMolecular Weight400,000 Da (18 chains)General DescriptionRat C1q is purified from pooled normal rat serum. C1q is part of the C1 complex, which is the first complement component in the classical pathway of complement. The C1 complex is a non-covalent assembly of three different proteins (C1q, C1r, and C1s) bound together in a calcium-dependent complex. C1q has six extended arms with domains at the end of each arm that bind to the Fc domains of immunoglobulins such as IgG or IgM. When antibodies bind toantigens, forming immune complexes, they cluster allowing two or more of the six C1q arms to bind to the Fc domains of antibodies. Rat IgG2 is very efficient when compared to IgG1 in activating complement (Medgyesi, G.A et., al., 1981). This is in contrast to the human system in which IgG1 activates complement but not IgG2 (Redpath, S. et. al., 1998). The binding of multiple arms of C1q to immune complexes causes the two C1r proteins in the complex (protease zymogens) to auto-activate. The activated C1r proteases cleave and activate the two C1s protease zymogens in the complex. The activated C1s cleaves complement component C4 releasing C4a and initiating covalent attachment of C4b to the activating surface. Activated C1s also cleaves C2 and the larger fragment of C2 binds to the surface-attached C4b forming C4b,C2a, the C3/C5 convertase of the classical pathway.Rat IgG1 cannot activate complement whereas rat IgG2 does.Physical Characteristics & StructureThe apparent molecular weight of rat C1q as determined by gel filtration has been reported to be 400,000 by Veerhuis, R. et al., (1985) and is calculated to be 420,000 based on its amino acid sequence. Rat C1q is a high molecular weight complex of 18 polypeptide chains. Each of the six arms of rat C1q contains three chains, an A chain (~30,000 daltons), a B chain (~28,000 daltons) and a C chain (~26,000 daltons) as determined by SDS/polyacrylamide gel electrophoresis (Wing, M.G. et al., (1993)).FunctionThe biological functions of C1q are described above in the General Description and Physical Characteristics sections.ApplicationsRat C1q can be used to coat ELISA plates to capture and quantitate immune complexes in samples from rat models used for studying immune complex related diseases and conditions.GeneticsNCBI Gene ID numbers for rat C1q are: C1q A chain (298566), C1q B chain (29687), and C1q C chain (362634). The genes for C1q chains A, B and C are all located on chromosome 5. The UniprotKB primary accession numbers for rat C1q are: C1q A chain (P31720), C1q B chain (P31721), and C1q C chain (P31722).Precautions/Toxicity/HazardsThis protein is purified from animal plasma/serum and therefore precautions appropriate for handling any animal blood-derived product must be used.ReferencesMedgyesi, G.A et., Miklos, K., Kulics, J., Fust, G., and Gergely, J. Bazin, H. (1981). Classes and subclasses of rat antibodies: reaction with the antigen and interaction of the complex with the complement system. Immunology 43, 171-176.Redpath, S., Michaelsen, T., Sandlie, I. and Clark, M. R. (1998). Activation of complement by human IgG1 and human IgG3 antibodies against the human leucocyte antigen CD52. Immunology 93, 595–600.Veerhuis, R., Van Es, L.A. and Daha, M.R. (1985). In vivo degradation of rat C1q induced by intravenous injection of soluble IgG aggregates. Immunology 54, 801-810.Wing, M.G., Seilly, D. J., Bridgman, D.J. and Harrison, R.A. (1993). Rapid isolation and biochemical characterization of rat C1 and C1q. Molecular Immunology 30, 433-440... Read More | Inquire | MAP kinase substrate (MBP) is a peptide substrate for ERK 1 and ERK 2 MAP kinases. Sequence contains amino acids 95-98 of myelin basic protein (MBP) including Thr 97 within the Pro-X-Ser/Thr-Pro MAP kinase substrate consensus sequence phosphorylation site.MAP kinase substrate (MBP)is a peptide MAP kinase substrate (MBP) is a peptide substrate for ERK 1 and ERK 2 MAP kinases. Sequence contains amino acids 95-98 of myelin basic protein (MBP) including Thr 97 within the Pro-X-Ser/Thr-Pro MAP kinase substrate consensus sequence phosphorylation site.MAP kinase substrate (MBP)is a peptide substrate for ERK 1 and ERK 2 MAP kinases... Read More |