| Description | CLCN3 Human Pre-designed siRNA Set A contains three designed siRNAs for CLCN3 gene (Human), as well as a negative control, a positive control, and a FAM-labeled negative control. Components CLCN3 siRNA-1: 5 nmol (HPLC) CLCN3 siRNA-2: 5 nmol (HPLC) CLCN3 siRNA-3: 5 nmol (HPLC) siRNA Negative Control:CLCN3 Human Pre-designed siRNA Set A contains three designed siRNAs for CLCN3 gene (Human), as well as a negative control, a positive control, and a FAM-labeled negative control. Components CLCN3 siRNA-1: 5 nmol (HPLC) CLCN3 siRNA-2: 5 nmol (HPLC) CLCN3 siRNA-3: 5 nmol (HPLC) siRNA Negative Control: 5 nmol (HPLC) FAM-labeled siRNA Negative Control: 5 nmol (HPLC) GAPDH siRNA Positive Control:5 nmol (HPLC)... Read More | Inquire | Product introduction：Aladdin ® SE is a kind of fluorescent dye with amino reactive activity. The SE group of these dyes can react with the amino group to produce a stable amide bond. Compared with other similar dyes on the market, aladdin ® is a new generation of fluorescent dyes Product introduction：Aladdin ® SE is a kind of fluorescent dye with amino reactive activity. The SE group of these dyes can react with the amino group to produce a stable amide bond. Compared with other similar dyes on the market, aladdin ® is a new generation of fluorescent dyes with stronger stability, better water solubility and better fluorescence intensity. Product parameters： Absmax/Em(nm)：648/664；Absmax/Em(nm)：0.03；Extinction coefficient(ε)：240000；Optimal DOL(IgG)：3-6； Usage:1. Experimental materials(1) IgG: IgG must not contain amine chemicals that can react with dyes, such as amino acids, Tris, BSA, gelatin, etc. If IgG contains such chemicals, PBS buffer with pH~7.4 should be used for pre dialysis treatment. The presence of azide compounds does not affect the labeling reaction.(2) Anhydrous DMSO(3) NaHCO3(4) Sephadex gel G-25 dialysis column(5) PBS buffer (pH~7.4)(6) NaN3(7) BSA2. Marking methods and steps(1) Prepare to label antibodiesDilute the antibody with 0.1 M NaHCO3 solution (pH~8.3) to a final concentration of 2.5 mg/mL. If the product is pre diluted with phosphate buffer, such as PBS buffer (without amino compounds), approximately 1/10 volume of 1M NaHCO3 mother liquor can be directly added to the buffer to achieve a final NaHCO3 concentration of 0.1 M.Note: When the protein concentration is 2.5 mg/mL, the labeling efficiency is approximately 35%. Protein concentrations below 2.5 mg/mL can also be used for labeling, but the labeling efficiency will decrease. When the protein concentration is higher than 5 mg/mL, the labeling efficiency may be higher. Due to differences in buffer and protein purity, more precise labeling efficiency is determined by practical operating conditions. If the protein concentration is too low, it can be concentrated by ultrafiltration.(2) Prepare dye storage solutionPreheat one tube at room temperature µ YF of Mole ® SE, add 0.1 mL of anhydrous DMSO to the tube, thoroughly vortex dissolve the dye, and prepare a dye storage solution with a concentration of 10 mM. If a trace amount of protein is used for labeling reactions, the dye needs to be diluted to a lower concentration.Note: a The remaining dye storage solution should be stored at a low temperature of -20 ℃ for future use. If anhydrous DMSO is used to prepare dye storage solution, the dye can be stored for at least one month.b. Dyes can also be prepared with deionized water, but due to the slow hydrolysis of dyes in water, it is best to prepare water based storage solutions for immediate use.(3) Mark reaction stepsa. Stir or vortex the protein solution, gradually adding 15-25 drops µ L dye storage solution (10 mM), with a molar ratio of dye/protein in the range of 9:1 to 15:1. YF ® Please refer to the table above for the range of DOL (number of dyes bound to each protein molecule) for SE labeled IgG antibodies.b. Stir the reaction at room temperature for 1 hour, and for trace labeling, shake and incubate on a shaker for 1 hour.Note: At the same time of the binding reaction, proceed to step 2 (4) to balance the dextran gel G-25 dialysis column.(4) Isolation of marker proteins from reaction solutiona. PBS buffer (pH~7.4) was used to balance the dextran gel G-25 dialysis column (10 mm × 300 mm).b. Add the reaction solution from step 3 (b) to the column and elute with 1 x PBS buffer.The first washed out chromophore is a dye protein complex.Note: a For small-scale labeling reactions, in order to avoid excessive dilution of the product, ultrafiltration devices can be used to remove free dyes from the complex.b. After the binding reaction is completed, if the dye protein complex is not separated in time, 50 can be added µ Terminate the reaction with L 1M lysine. In most cases, this operation is not necessary because the remaining unreacted dyes have been fully hydrolyzed at the end of the reaction.3. Determine DOL(1) The determination of protein concentration and antibody concentration can be calculated using the following formula:C (mg/mL)={[A280- (Amax x x Cf)]/1.4} x dilution factor;a. C refers to the concentration of antibodies collected in the experiment;b. Dilution factor refers to the dilution factor used in photometric measurements;c. A280 and Amax refer to the absorbance at 280 nm and the absorbance at the absorption wavelength, respectively;d. Cf is the correction factor, YF ® Please refer to the table above for the Cf value of SE dyes;Note: The protein solution eluted through the column may have a high concentration when used directly for absorbance detection, so it needs to be diluted to approximately 0.1 mg/mL. The dilution factor (i.e. dilution factor) needs to be determined from the initial number of antibodies (e.g. 5 mg) and the overall elution of protein solutionEstimate based on the product.(2) Estimation of DOLDOL is calculated using the following equation:DOL=(Amax x x Mwt x Dilution Factor)/（ ε X C)a. Amax, dilution factor, C value has been clearly defined in 3 (1);b. Mwt refers to the molecular weight of IgG (150000);C. c ε It's YF ® The molar absorption coefficient of SE, refer to the table on the first page;d. Mark YF ® The optimal DOL value for SE IgG antibodies can be found in the table on the first page. Although DOL values may fluctuate, good experimental results can also be achieved.Matters needing attention：1. if the labeled protein needs long-term storage, it is recommended to add 5-10 mg/ml BSA and 0.01-0.03% NaN3 to prevent protein denaturation and microbial breeding. Store at 4 ℃ away from light. If glycerol with a final concentration of 50% is added, it can be stored at -20 ℃. It can be stably stored for more than one year. 2. keep away from light during operation. The mixing speed should be appropriate to avoid bubbles. 3. when installing the chromatographic column, try to make the column body uniform, the column surface flat, and free of bubbles and cracks. 4. pay attention to adding the sample when the column top buffer is tangent to the gel plane. When eluting, add the eluent when the sample is tangent to the gel plane. 5. other factors affecting the labeling efficiency also include temperature, reaction time, pH, the amount of fluorescent dye and protein, etc., which should be controlled. 6. for your safety and health, please wear laboratory clothes and disposable gloves.Scope of application：Protein nucleic acid labeling dye... Read More | Purity>90% by SDS-PAGEExtinction Coeff.A280 nm = 0.988 at 1.0 mg/mLPrecautionsUse normal precautions for handling human blood productsGeneral DescriptionNative human C9 is a naturally glycosylated (7.8%) protein composed of a singlepolypeptide chain. The molecular weight is 71,000 Da. C9 binds toPurity>90% by SDS-PAGEExtinction Coeff.A280 nm = 0.988 at 1.0 mg/mLPrecautionsUse normal precautions for handling human blood productsGeneral DescriptionNative human C9 is a naturally glycosylated (7.8%) protein composed of a singlepolypeptide chain. The molecular weight is 71,000 Da. C9 binds to the C5b-8 complex and forms the mature membrane attack complex (MAC) on cell membranes. Each pathway of complement activation generates proteolytic enzyme complexes (C3/C5 convertases) which are bound to the target surface (Ross, G.D. (1986)). These enzymes cleave a peptide bond in the larger alpha chain of C5 releasing the anaphylatoxin C5a and activating C5b. This is the only proteolytic step in the assembly of the C5b-9 complex. C5b is unstable, but it remains bound to the activating complex for a brief time (~2 min) during which it either binds a single C6 from the surrounding fluid or decays and is no longer capable of forming MAC. The C5b,6 complex may also remain bound to the C3/C5 convertase where the binding of a single C7 exposes a membrane-binding region and C5b,6,7 can partially insert into the bilipid layer of the target cell. Up to this point the complex may diffuse away from the target cell and enter the membrane of a nearby cell. This is called bystander lysis or “reactive lysis” and can be a significant source of pathology. Each C5b-7 complex can bind one C8 protein molecule which results in the complex inserting more firmly into the membrane. The C5b-8 complex is capable of causing lysis without C9 although this is slow and requires many more complexes per cell than with C9. This property complicates C9 titrations since the precursor (C5b-8) can also cause lysis. The primary role of C8 is to catalyze the binding of C9 and each bound C9 can bind another C9 initiating formation of a ring structure containing up to 18 molecules of C9 (Podack, E.R. (1984)). C5b-9 complexes with one or more C9 are referred to as the Membrane Attack Complex (MAC) of complement. Not all C5b-8 complexes have complete rings of C9 with the average being only three C9 per C5b-8complex. Nevertheless, these structures are capable of causing lysis if enough are formed in a given cell. Completed protein rings of C9 form the pores seen on electron micrographs and they result in leakage of metabolites and small proteins out of the cell as well as movement of water into the cell. If sufficient numbers are inserted into a cell membrane then water flowing into the cell, due to osmotic pressure, will rupture the cell membrane allowing the entire contents of the target cell (or a bystander cell) to be released. Either process may result in cell death. Originally it was thought that this required only one C5b-9 complex per cell (referred to as the “one hit theory” of lysis (Rommel F.A. and Mayer, M.M. (1973)), but this is probably not correct. For example, an erythrocyte without CD59 requires ~850 C5b-9 complexes, as measured by the number of C7 molecules, for lysis to occur (Bauer, J. et al. (1979)). Host cells protected from MAC by CD59 require sufficient numbers of C5b-9 to tie up all the CD59 and then ~850 C5b-9 in addition. Lysis of nucleated cells requires many more C5b-9 complexes due to their size and due to the presence of multiple defense mechanisms in such cells.Physical Characteristics & StructureThe molecular weight of C9 is 71,000 Da and it is a single polypeptide chain. The protein contains 7.8% carbohydrate attached at two N-linked glycosylation sites. The pI of C9 is 4.7. C9 may polymerize spontaneously forming MAC rings without C5b-8. The rings formed from pure C9 as well as the completed rings formed by C5b-9 with 12 to 18 C9 molecules have the unusual property of being stable in boiling SDS even though they are non-covalently bound. Function See General Description above. Assays Assays for C9 function are complicated by the fact that if excess C5-C8 is used cells (EA or Er) will be lysed by the C5b-8 complex. Thus it is critical to use limited C8 in these assays to keep the background lysis to a minimum. The simplest assay for C9 is to use C9-depleted human serum and measure the lysis of EA (classical pathway) or Er (alternative pathway) as a function of the concentration of added test sample or standard purified C9. Each unique application might require appropriate conditions to be determined. However, a typical assay would involve mixing on wet ice ~5 µL C9-Dpl, C9-containing sample diluted with GVB⁺⁺ to contain from 1 to 10 ng C9, and sufficient GVB⁺⁺ to bring the volume to 300 µL. EA (3 X 10⁷ cells in 200 µL) diluted in GVB⁺⁺ should be added last. Purified C9 or normal human serum (NHS) may be used as a source of C9. The reaction mixture is incubated for 30 min at 37℃ and 1 mL of cold GVBE added, mixed and centrifuged to spin down unlysed cells. The released hemoglobin in the supernatant is then analyzed at 415 nm and compared to blanks without C9 (background lysis control) and cells incubated with 275 µL water instead of GVB⁺⁺ and 25 µL C9-Dpl (100% lysis control). Note as mentioned above, at inputs of serum higher than ~5 µL of C9-Dpl, EA and other target cells may also be lysed in the absence of C9 depending on the cells’ susceptibility to C5b-9.Many other assays have been described using EA preloaded with C1 (EAC1 cells) or preloaded with the classical pathway C5 convertase (EAC1423 cells), however, all these assays require the use of multiple purified complement components or more difficult-to-prepare reagents (Dodds, A.W. and Sim, R.B. (1997; Morgan, B.P. (2000);Tack, B.F., et al. (1981)).ApplicationsSee General Description aboveIn vivoThe normal serum concentration of C9 is 60 µg/mL (normal range 47 to 70µg/mL). The primary site of synthesis is the liver. C9 is also produced by monocytes, macrophages, fibroblasts and glial cells. C9 is an acute phase protein and its synthesis is stimulated by cytokines (such as IFNγ) that stimulate increased biosynthesis of many other complement proteins.RegulationMany proteins and other components of plasma have an inhibitory effect on the lytic activity of C5b-9 complexes but there are no specific C9 inactivators. Most of the C5b-9 inhibitors interact with the complex after the C5b-7 stage. If any of the C5bcontaining complexes fail to insert into a membrane they may self-aggregate or bind to regulatory proteins the most prevalent of which is S Protein. S Protein (also called vitronectin) is an 80,000 Da plasma protein found bound to most soluble C5b-9 complexes. Many other serum components inhibit or partially inhibit lysis by C5b-9 and these include SP40,40 (also known as clusterin and apolipoprotein J) and many plasma lipoprotein complexes (LDL, HDL, etc.).Host cells protect themselves from C5b-9 by a variety of mechanisms. Membrane proteins DAF, MCP, and CR1 inhibit formation of C3/C5 convertases preventing MAC formation. CD59, also called “homologous restriction factor” and “protectin”, is a 18,000 to 20,000 Da ubiquitous component of cell membranes that is very effective at binding to and inhibiting the lytic potential of C5b-8 and C5b-9 complexes. The speciesspecificity of CD59 is not absolute and many mammalian CD59 proteins inhibit or partially inhibit MAC from other species. The specificity that is observed appears to be due to incompatibilities between C8 of one animal and the CD59 of another. Like DAF, CD59 contains a GPI anchor (a post-translationally added lipid tail that inserts into the bilipid layer of the cell). The disease PNH is caused by the loss of enzymes that attach the GPI tail, thus depriving cells of the ability to express DAF and inactivate C3/C5 convertases and the ability express CD59 to inactivate C5b-9. This results in the spontaneous lysis by complement of the most susceptible cells such as erythrocytes and platelets.GeneticsHuman chromosome location 5p 13. Accession number HSC6A. Mouse chromosome 15. Human genomic structure: the gene spans 100 kb with 11 exons.DeficienciesHuman C9 deficiencies are quite common. A well documented study found that 1:1000 people in the Janaese population were C9 deficient although other ethnic groups have lower incidents of C9 deficiency (Horiuchi, T. et al. (1998)). Deficiencies exhibit autosomal recessive transmission. Patients generally exhibit abnormally high susceptibility to recurrent meningococcal meningitis and systemic neisserial infections. Partial deficiencies do not seem to show adverse clinical effects.DiseasesSee Deficiencies above.Precautions/Toxicity/HazardsThis protein is purified from human plasma, therefore precautions appropriate for handling any blood-derived product must be used even though the source was shown bycertified tests to be negative for HBsAg, HTLV-I/II, STS, and for antibodies to HCV, HIV-1 and HIV-II.Hazard Code: B WGK Germany 3MSDS available upon request... Read More | Purity>95% (SDS-PAGE&HPLC) Endotoxin level<0.1 EU/µgFunctionMay regulate apoptosis, cell proliferation and cell differentiation. Binds beta-galactoside and a wide array of complex carbohydrates. Inhibits CD45 protein phosphatase activity and therefore the dephosphorylation of Lyn Purity>95% (SDS-PAGE&HPLC) Endotoxin level<0.1 EU/µgFunctionMay regulate apoptosis, cell proliferation and cell differentiation. Binds beta-galactoside and a wide array of complex carbohydrates. Inhibits CD45 protein phosphatase activity and therefore the dephosphorylation of Lyn kinase.Gal-1 is also engaged in many protein-protein interactions. Gal-1 plays a number of crucial roles in neuronal cell differentiation and survival in both the central and the peripheral nervous systems, and the establishment and maintenance of T-cell tolerance and homeostasis in vivo... Read More |