| Description | Aladdin's Anti-V5 Affinity Gel, also known as Anti-V5 IP Gel, Anti-V5 immunoprecipitation gel or Anti-V5 agarose gel, is agarose gel covalently coupled with high-quality mouse monoclonal antibody that recognizes the V5 tag sequence (GKPIPNPLLGLDST). This product can specifically bind V5-tagged Aladdin's Anti-V5 Affinity Gel, also known as Anti-V5 IP Gel, Anti-V5 immunoprecipitation gel or Anti-V5 agarose gel, is agarose gel covalently coupled with high-quality mouse monoclonal antibody that recognizes the V5 tag sequence (GKPIPNPLLGLDST). This product can specifically bind V5-tagged proteins expressed in animals, plants, and microorganisms, and can be used for immunoprecipitation (IP) and purification of V5-tag fusion proteins or their protein complexes.V5-tag is a peptide consisting of 14 amino acids (GKPIPNPLLGLDST) isolated from monkey parainfluenza virus V5, which can be fused either to the N-terminus or the C-terminus of a target protein to facilitate protein purification and immunoassays. V5-tag usually does not interact with the target protein, and in most cases does not affect the function of the target protein; With Aladdin's V5 antibodies, Anti-V5 magnetic beads, or Anti-V5 affinity gel, V5-tagged proteins can be used for examination of protein expression level and subcellular localization, protein purification, and immunoassays.This product has high binding capacity and strong specificityPrecautions：This product must be fully resuspended by inverting the tube several times prior to use.This product contains a small amount of preservative, which does not affect routine IP assays and protein purification. But for special experiments that might be interfered by the preservative, the gel should be washed 3 times with appropriate solutions such as TBS prior to use.For immunoprecipitation assays, it is recommended to include both positive and negative controls. Protein samples should be purified as soon as possible after collection and should always be placed at 4℃ or on ice to minimize protein degradation or denaturation. Protein degradation can be inhibited by adding appropriate protease inhibitors, such as Protease Inhibitor Cocktail for General Use, Protease and Phosphatase Inhibitor Cocktail for General Use (MS-safe, 50X), Protease Inhibitor Cocktail for Mammalian Cell and Tissue Extracts, Protease and Phosphatase Inhibitor Cocktail for Mammalian Cell and Tissue Extracts, etc.High concentrations of DTT, mercaptoethanol, guanidine hydrochloride, etc. may have a certain effect on the binding of this product to V5-tagged proteins, but Cell lysis buffer for Western and IP, RIPA Lysis Buffer or NP-40 Lysis Buffer are applicable to this product. For the main features and differences of various lysis buffers produced by and the selection of lysis buffers, please refer to the website at http://www.aladdin-e.com/support/lysis-buffer.htm.If the insoluble substance in protein samples can not be removed completely by centrifugation, samples can be filtered with a 0.45µm filter before performing the assay.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1.Preparation of protein samplesa.Lysis cells or tissues with appropriate lysis buffer, such as ’s Cell Lysis Buffer for Western and IP. Under certain circumstances, ’s RIPA Strong Lysis Buffer, RIPA Medium Lysis Buffer, or RIPA Weak Lysis Buffer can also be used. Other lysis buffers with a pH6-8 can also be used.b.After lysis and centrifuge, keep the supernatant at 4℃ or on ice for subsequent use. We recommend performing the subsequent procedures on the same day. Otherwise, make aliquots and store them at -80℃ for future use.2.Preparation of Anti-V5 Affinity GelAs the Anti-V5 Affinity Gel is stored in a protective solution containing 50% glycerol, it needs to be washed properly before adding protein samples.a.Gently resuspend the Anti-V5 Affinity Gel to be homogeneous. Transfer 20µl of gel suspension per 100µl of protein sample into a clean centrifuge tube. Note: It is easier to aspirate the gel suspension using a large aperture pipette tip (e.g., by cutting off part of the tip with scissors).b.Add 1X TBS to a final volume of 0.5ml, and gently resuspend the Anti-V5 Affinity Gel by pipetting or vortex. Centrifuge at 6000×g for 30 seconds at 4℃, remove the supernatant carefully without disturbing the gel. Repeat this step twice.c.Resuspend the Anti-V5 Affinity Gel with 1X TBS equal to the initial volume of gel suspension (e.g., if 20µl of gel suspension is taken, add 20µl of 1X TBS).3.Protein binding1)Add 20µl of washed Anti-V5 Affinity Gel per 100µl of protein sample, mix well, and incubate at 4℃ for 1-2 hours or overnight with gentle shaking on a rotary mixer.2)After incubation, centrifuge at 6000×g for 30sec at 4℃ and remove the supernatant carefully without disturbing the gel. Note: A portion of supernatant can be reserved in a clean microfuge tube for examination of the binding results.3)Gently resuspend the Anti-V5 Affinity Gel in 500µl of 1X TBS and place in an ice bath on a shaker for 5 minutes. Centrifuge at 6000×g for 30 seconds at 4℃ and carefully remove the supernatant. Repeat the wash thrice, then keep the sample on ice for elution. 4.ElutionBased on the features of the target protein or downstream applications, one of the following three elution methods can be used.1)Competitive elution with the V5 peptide. This is a non-denaturing elution method with a high elution efficiency, and the eluted proteins do not contain the light and heavy chains of V5 antibody.a.Preparation of V5 peptide elution buffer: Dissolve the V5 Peptide (, P9813) in 1X TBS buffer to a final concentration of 150µg/ml, or dilute the 5mg/ml V5 peptide solution with 1X TBS buffer to a final concentration of 150µg/ml. b.For every 20µl of initial gel volume, add 100µl of V5 Peptide elution buffer (150µg/ml). Resuspend the gel gently and incubate for 30-60 minutes at room temperature on a rotary mixer, or 1-2 hours at 4℃. To improve the elution efficiency, increase the incubation time or repeat the elution. c.After incubation, centrifuge at 6000×g for 30 seconds at 4℃, and transfer the supernatant to a new microfuge tube. The supernatant contains the V5-tagged protein and its protein complex.d.Store the supernatant at 4℃ for immediate use, or -20℃/ -80℃ for long-term storage. 2)Acidic elution: This method is non-denaturing, relatively fast and efficient. The eluted proteins also retain their original biological activity, which facilitates subsequent analysis and detection.a.Preparation of acidic elution buffer (0.1M Glycine-HCl, pH3.0) and neutralization buffer (0.5M Tris-HCl, pH7.4, 1.5M NaCl). b.For every 20µl of initial gel volume, add 100µl of acidic elution buffer. Resuspend the agarose gently and incubate for 5 minutes at room temperature on a rotary mixer.Note: The incubation time should not exceed 15 minutes.c.After incubation, centrifuge at 6000×g for 30 seconds at 4℃, and transfer the supernatant to a new microfuge tube. Immediately add 10µl of neutralization buffer and mix well. d.To achieve the maximum elution efficiency, repeat steps b-c and combine the supernatant containing the V5-tagged protein and its protein complex. e.Store the supernatant at 4℃ for or immediate use, or -20℃/-80℃ for long-term storage. Note 1: The elution efficiency of the acidic elution method might be lower than the other two elution methods.Note 2: The elution efficiency of the acidic elution method depends on characteristics of the target protein. To obtain a higher elution efficiency, the pH of the acidic elution buffer can be optimized from 2.5 to 3.1. The pH or amount of neutralization buffer needed to neutralize the eluates should also be adjusted appropriately.3)Elution with the 1X SDS-PAGE loading buffer: This method is a denaturation method, and the obtained proteins are suitable for SDS-PAGE electrophoresis or WB blot analysis.a.Preparation of SDS-PAGE loading buffer: We recommend using ’s SDS-PAGE Sample Loading Buffer (2X). Usually, the SDS-PAGE protein loading buffer contains a reducing agent such as DTT, and the eluted protein sample will contain the light chain and heavy chain of the V5 antibody.b.For every 20µl of initial gel volume, add 20µl of 2X SDS-PAGE sample loading buffer, mix well and heat at 95℃ for 5 minutes. c.Centrifuge at 6000×g or 4℃ for 30 seconds. Take the supernatant for SDS-PAGE electrophoresis or Western blot analysisNote: The Agarose cannot be reused because SDS in the loading buffer destroys V5 antibodies.FAQ: Problem Possible Causes Solution Large amount of tagged protein found in the flow through. Binding time is not enough. If using batch method, increase the binding time experimentally; If using column method, use a lower flow rate when loading samples. Column is overloaded. Reduce the amount of the sample added to the gel or increase the amount of gel. Tag is not accessible to gel. Expose the epitope tag by adding low amount of denaturant to the protein extract (dialysis may be needed before applying onto gel), or fuse thetag to the other terminus of the target protein. Gel has not been regenerated since last purification. Perform gel regeneration procedure prior to binding. Reagent compatibility problem. Dialyze the sample against TBS before purification procedure. The target protein has been degraded. 1.Prepare fresh lysates. Avoid using frozen lysates.2.Perform purification at lower temperature, such as 4℃.3.Use appropriate protease inhibitors in the lysate or increase their concentrations to prevent degradation of the fusion protein. Very few or notagged protein exists in the eluate. Protein is not completely eluted. Change elution methods. No target protein expressed. Make sure the protein of interest contains the tag by Western blot or dot blot analyses. Very low protein expression level. 1.Use larger volume of cell lysate.2.Optimize expression conditions to raise the protein expression level. Washes are too stringent. 1.Reduce the number of washes.2.Avoid adding high concentrations of NaCl to the mixture.3.Use solutions that contain less or no detergent Incubation times are inadequate. Increase the incubation times with the affinity gel (from several hours to overnight). Interfering substance is present in sample. 1.Lysates containing high concentrations of DTT, 2-mercaptoethanol, or other reducing agents may destroy antibody function, and must be avoided.2.Excessive detergent concentrations may interfere with the antibody-antigen interaction. Detergent levels in buffers may be reduced by dilution. Detection system is inadequate. If Western blot detection is used:1.Check primary and secondary antibodies using proper controls to confirm binding and reactivity.2.Verify that the transfer was adequate by staining the membrane with Ponceau S.3.Use fresh detection substrate or try a different detection system. Multiple protein bands found in the eluate. The protein is not stable at room temperature. Purify the target protein at lower temperature, such as 4℃. Protein degradation due to proteases activity during purification process. Add protease inhibitors to cell lysate. Non-specific binding. 1.Prepare cell lysate again.2.Add additional wash steps. Background is too high. Proteins bind nonspecifically to the monoclonal antibody, the gel beads, or the microcentrifuge tubes. 1.Pre-clear lysate with Mouse IgG-Agarose to remove nonspecific binding proteins.2.After suspending beads for the final wash, transfer entire sample to a clean microcentrifuge tube before centrifugation. Washes are insufficient. 1.Increase the number of washes.2.Prolong duration of the washes, incubating each wash for at least 15 minutes.3.Increase the salt and/or detergent concentrations in the wash solutions.4.Centrifuge at lower speed to avoid nonspecific trapping of denatured proteins from the lysate during the initial centrifugation of the affinity gel complexes... Read More | Protein Purity>90 % by SDS PAGEExtinction CoeffA280 nm = 0.725 at 1.0 mg/mL for pure C1s-C1INH ComplexMolecular Weight196,000 Da (1 chain)General DescriptionThe product C1s-C1INH Complex is made by interacting purified protease inhibitor C1-INH with purified C1s enzyme followed by purification. Protein Purity>90 % by SDS PAGEExtinction CoeffA280 nm = 0.725 at 1.0 mg/mL for pure C1s-C1INH ComplexMolecular Weight196,000 Da (1 chain)General DescriptionThe product C1s-C1INH Complex is made by interacting purified protease inhibitor C1-INH with purified C1s enzyme followed by purification. The protease inhibitor C1-INH prevents the spontaneous activation of complement and limits consumption of C2 and C4 by rapidly inactivating C1r, C1s and MASP2. It is the only plasma serine protease inhibitor (Serpin) capable of interacting with and inhibiting activated C1. C1-INH interacts with the catalytic sites of both C1r and C1s. The interaction with activated C1r and C1s is covalent resulting in complexes which are stable to SDS. C1s and C1r enzymes, however, are irreversibly inactivated by binding to C1-INH. C1s-C1INH is a very stable complex that remains intact even when subjected to freeze/thaw cycles with almost no loss of the complex form.Physical Characteristics & StructureThe C1s enzyme-C1INH complex is composed of two disulfide linked chains from C1s enzyme (A chain 58,000 Da and B chain 28,000 Da) and one covalently linked chain from C1-INH (75,000 Da).SDS-PAGE analysis of the C1s-C1INH complex shows a single band of about 161,000 Da under nonreducing conditions. Under reducing conditions, the C1s-C1INH complex exhibits two bands: A 58,000 Da band corresponding to the A chain of C1s enzyme and a second 103,000 Da band resulting from C1INH (75,000 Da) covalently bond to the B chain (28,000 Da) of C1s enzyme.RegulationActivated C1s is controlled by C1-INH. C1s enzyme and C1-INH form a covalent complex that is resistant to separation on SDS gels. During complement activation C1 complex is rapidly activated by binding to immune complexes. The resulting activated C1s and C1r are rapidly inactivated by interaction with C1-INH (Ziccardi, R.J. (1982)). Binding to immune complexes is fast (10-20 sec) and activation of the bound C1 complex takes several minutes, but C1-INH has also been shown to be fast and no active C1r or C1s remain 4 min after addition of immune complexes to plasma (Ross, G.D. (1986); Ziccardi,R.J. (1981)). The binding of C1-INH to activated C1 releases both C1r and C1s from the complex leaving C1q bound to the immune complex. The released complexes contain four molecules: C1-INH-C1r-C1s-C1-INH. The reaction of C1 esterase inhibitor with activated C1 is very fast with the estimated half-life of C1r and C1s being approximately 15 seconds in serum. In fact, at serum concentrations of C1- INH little or no additional C4 or C2 activation occurs 3 min after immune complexes are added because all the C1r and C1s molecules have been inactivated and removed from the C1q which remains bound to the immune complex (Ross, G.D. (1986); Morley, B.J. and Walport, M.J. (2000); Rother, K., et al. (1998); Ziccardi, R.J. (1982a and 1982b); Morgan, B.P. (1990)). The interaction of purified C1s enzyme and C1-INH is slower.FunctionSee General Description and Regulation above.ApplicationsC1s-C1INH complex can be used in studies designed for developing and identifying inhibitors of C1s-C1INH complex formation and thus lead to the possible development of therapeutics for inhibiting complement activation via the classical pathway.GeneticsThe EMBL/Genbank cDNA accession number for C1s is J04080. The gene for C1s is located on chromosome 12p13. The EMBL/Genbank cDNA accession numbers for C1-INH are M13656 and X54486 (human) and Y10386 (mouse). The gene for C1-INH is located on chromosome 11p11.2-13. DeficienciesC1s deficient patients are prone to systemic lupus erythematosus (SLE) and recurrent pyogenic infections (Rother, K., et al. (1998)). They lack classical pathway function. The genetic disorder hereditary angioedema (HAE) is caused by a partial deficiency of C1-INH. Patients with HAE have low functional C1-INH levels in blood and have recurrent episodes of systemic or localized edema.DiseasesSee section titled Deficiencies above. Precautions/Toxicity/HazardsThis protein is purified from human serum and therefore precautions appropriate for handling any blood-derived product must be used even though the source was shown by certified tests to be negative for HBsAg, HTLV-I/II, STS, and for antibodies to HCV, HIV-1 and HIV-II.ReferencesZiccardi, RJ. (1982) A new role for C-1-inhibitor in homeostasis: control of activation of the first component of human complement. J. Immunol. 128:2505-2508.Ross, G.D. (1986) Immunobiology of the Complement System. (ISBN 0-12-5976402) Academic Press, Orlando.Ziccardi, R.J. (1981) Activation of the early components of the classical complement pathway under physiologic conditions. J. Immunol. 126:1769-1773.Morley, B.J. and Walport, M.J. (2000) The Complement Facts Book. (ISBN 0127333606) Academic Press, London.Rother, K., Till, G.O., and Hӓnsch, G.M. (1998) The Complement System. (ISBN 3-540- 61894-5) Springer-Verlag, Heidelberg.Ziccardi, R.J. (1982a) Spontaneous activation of the first component of human complement (C1) by an intramolecular autocatalytic mechanism. J. Immunol. 128:2500- 2504.Ziccardi, RJ. (1982b) A new role for C-1-inhibitor in homeostasis: control of activation of the first component of human complement. J. Immunol. 128:2505-2508. Morgan, B.P. (1990) Complement Clinical Aspects and Relevance to Disease. (ISBN 0- 12-506955-3) Academic Press, London... Read More | Protein Purity>97% by SDS-PAGEExtinction Coeff.A280 nm = 0.41 at 1.0 mg/mlMolecular Weight10,400 Da (single chain)General DescriptionNatural human C5a is prepared from human C5 protein by cleavage of the peptide bond between C5a and C5b by the human C5 convertase. C5a is a naturally glycosylated Protein Purity>97% by SDS-PAGEExtinction Coeff.A280 nm = 0.41 at 1.0 mg/mlMolecular Weight10,400 Da (single chain)General DescriptionNatural human C5a is prepared from human C5 protein by cleavage of the peptide bond between C5a and C5b by the human C5 convertase. C5a is a naturally glycosylated polypeptide containing 74 amino acids with a molecular weight of approx. 10,400 daltons. Itcontains 25% carbohydrate attached to a single Asn residue at position 64. This carbohydrate is of variable structure leading to a broad distribution of MW upon analysis by mass spectroscopy. C5a is the most potent anaplylatoxin (compared to C3a and C4a). Its biological properties include being strongly chemotactic for neutrophils (PMN), causing smooth muscle contraction, increasing vascular permeability, causing histamine and TNFalpha release, and causing lysosomal degranulation of immune cells. C5a acts through the C5a Receptor (C5aR, CD88, a G-protein coupled receptor) on PMN, monocytes, alveolar macrophages, and mast cells. A second receptor of unknown function (C5L2, gpr77) has been identified. Due to the widespread expression of C5a receptors and the results from C5aR KO mice it is believed that C5a and its receptors have many non-immunolgical functions in organ development, CNS development, neurodegeneration, tissue regeneration and hematopoiesis (Monk, P.N. et al. (2007)).Native versus Recombinant C5aNumerous recombinant forms of C5a are sold by many companies. In side-by-side biological testing, we have found that our native C5a is 10- to 100-fold more active per µg than all but one of these recombinant proteins. Structurally not a single one of the recombinant proteins on the market has the correct amino acid sequence or structure. They have extra amino acids at the N-terminal (such as 6 His tags), different amino acids in the sequence itself (some were produced from the original, but incorrect amino acid sequence), and none possess the 25% carbohydrate at Asn 64. In fact, one recombinant C5a on the market has approximately 30 additional amino acids at the N-terminal end due to the cloning vector used. This is a 40% addition of nonsense structure to the C5a molecule. Both our C5a and our C5adesArg are native proteins produced by the native human C5 convertase. Physical Characteristics & StructureMolecular weight: 10,400 (+ 1,000 due to variable glycosylation)Deglycosylated MW: 8,271 (observed). Calculated monoisotopic mass 8268;Calculated average mass 8273.Isoelectric point: pI = 8.9Carbohydrate content: ~25% carbohydrate (heterogeneous) Amino acid sequence: TLQKKIEEIA AKYKHSVVKK CCYDGACVNNDETCEQRAAR ISLGPRCIKA FTECCVVASQ LRANISHKDM QLGRCAS Number: 80295-54-1MDL Number: MFCD00130842NMRderived structure: FEBS Lett. 238:289-294, 1988; Biochemistry 28:172-185,1989; Biochemistry 29:2895-2905, 1990; Proteins 28:261-267, 1997.FunctionC5a released from C5 by C5 convertases initiates a multitude of inflammatory reactions. C5a causes neutrophils to become adherent to endothelium and to migrate to the site of complement activation by chemotaxis where it stimulates release of PMN granule contents and reactive oxygen species. The biological properties of C5a include being strongly chemotactic for neutrophils (PMN), causing smooth muscle contraction, increasingvascular permeability, causing histamine release, and initiating lysosomal degranulation of a variety of immune cells. C5a acts through the C5a Receptor (C5aR, a G-protein coupled receptor) on PMN, monocytes, alveolar macrophages, dendritic cells, mast cells, glial cells and smooth muscle cells. Rapid release of C5a and other anaphylatoxins can cause systemic effects as well as local changes. Anaphylatic shock is a generalized circulatory collapse similar to that caused by an allergic reaction and is caused by C3a and C5a which are generally released together. AssaysThe multitude of biological functions of C5a has resulted in the use of many different assay systems (Dodds, A.W. and Sim, R.B. (1997)). The most typical biological assays being smooth muscle contraction assays using guinea pig ileum, chemotaxis assays using neutrophils or granule-release assays using human PMN or similar cell lines. Granule release is generally followed by measuring the release of myeloperoxidase. In addition, assays have been described that measure ATP release from guinea pig platelets, serotonin relaease from guinea pig platelets, N-acetyl-beta-D-glucosamidase release from differentiated U937 cells and calcium release from differentiated U937 cells. These assays have been described in detail (Dodds, A.W. and Sim, R.B. (1997)). Functional responses have been detected in the sub-picomolar concentration range for purified human C5a (Gerard, C. et al. (1981); Hugli, T.E. et al. (1981)).ELISA kits for the assay of C5a levels (or more correctly C5a desArg levels) in blood and other fluids are sold by many companies. A radioimmunoassay for C5a/C5a desArg is also available. These measurements are useful for detecting complement activation in vivo, but the interpretation of their meaning is complicated by the fact that clearance of the anaphylatoxins is rapid. In vivoThe resting serum concentration has been reported to be approximately 4 nMalthough it is difficult to draw or store blood without 1 to 10 % C5 activation (Watkins, J.(1987)). The presence of EDTA and Futhan in the collection tubes can minimize this background. Full activation of all C5 in blood (75 µg/mL) would result in ~380 nM C5a(~3.9 µg/mL). Due to the extreme sensitivity of many C5a responses, a response can theoretically be initiated by activation of approximately one millionth of the C5 in a local area.RegulationC5a levels are regulated by three processes: formation, inactivation and clearance. The enzymes that cleave C5 and release C5a (collectively called C5 convertases) do so at very slow rates. Operating at Vmax the best enzymes only cleave one C5 every three minutes (Rawal, N. and Pangburn, M.K. (2001)). C5a is “inactivated” by removal of its Cterminal arginine amino acid. The product C5a desArg (or C5a without the C-terminal arginine) is produced by the action of the plasma enzyme carboxypeptidase N (Mueller-Ortiz S.L. et al. (2009)). This inactivation is rapid and most C5a is converted to C5a desArg within minutes of its formation. “Inactivated” C5a still possesses approx. 1% of its anaphylatoxic and chemotatic activities, but its stimulatory activity is only reduced 10-fold. Thus, C5a desArg retains considerable biological activity even though it is frequently called inactivated C5a. Because of the large number of cells bearing C5a receptors (endothelial, immune, smooth muscle, neuronal, etc.) the capture, internalization and digestion of C5a results in its rapid removal from circulation.DeficienciesA deficiency of C5 or a deficiency of the enzymes that cleave C5 to generate C5a result in the absence of C5a. There are no known complete deficiencies of C5 convertases. Examples of C5 deficient humans and mice exist. In fact, many laboratory mouse strains in common use were shown to have been bred with a deficiency of C5 (A/HeJ, AKR/J, DBA/2J, NZB/B1NJ, SWR/J, and B10.D2/nSnJ). The lack of C5 prevents formation of the membrane attack complex of complement and precludes formation of C5a. Humans lacking C5 are susceptible to repeated infections from a wide variety of organisms, primarily gramnegative bacteria. Meningococcal and gonococcal neisserial infections are especially problematic. The degree to which pathologies associated with C5 deficiency are due to the lack of C5 or the absence of C5a is unclear, but information on this is being acquired from receptor knock-out animals.DiseasesSee Deficiencies above.Precautions/Toxicity/HazardsThis protein is purified from human serum and therefore precautions appropriate for handling any blood-derived product must be used even though the source was shown by certified tests to be negative for HBsAg, HTLV-I/II, STS, and for antibodies to HCV, HIV-1 and HIV-II.Injection can cause anaphylatic shock which is a generalized circulatory collapse similar to that caused by an allergic reaction.Hazard Code: B WGK Germany 3... Read More | Product contentF665774Component5 mLStorageF665774A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665774B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.F665774CddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a preProduct contentF665774Component5 mLStorageF665774A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665774B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.F665774CddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a pre-mixed system for real-time fluorescence PCR by probe method (TaqMan, Molecular Beacon, etc.), with a concentration of 2×, including Fast Taq DNA Polymerase, PCR Buffer, dNTPs, Mg2+ and so on, which is easy and convenient to operate. It is mainly used for the detection of genomic DNA target sequence and cDNA target sequence after RNA reverse transcription. The Fast Taq DNA Polymerase contained in this product can effectively reduce the non-specific amplification generated by the non-specific binding of primers and templates or primer dimerization at room temperature, and the activation of the enzyme only needs to be incubated at 95 ℃ for 30 s. The whole PCR reaction process can save about 40 minutes compared with the ordinary reaction, which greatly shortens the reaction time of PCR. The combination of unique PCR buffer system and fast hot start enzyme effectively inhibits the generation of non-specific products and significantly improves the PCR amplification efficiency with stronger fluorescence signal, higher sensitivity and wider linear range. The product has a wide range of applications and can be used for both normal and rapid quantitative PCR programs.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration (F665766):Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.Instruments that require Low ROX calibration (F665768):ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 system. Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and more.Instruments that require High ROX calibration (F665774):ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention1. Before use, please mix gently by turning up and down, avoid foaming as much as possible, and use after brief centrifugation.2. Avoid repeated freezing and thawing of this product, repeated freezing and thawing may degrade the product performance. This product can be stored for long term at -20℃, protected from light. If frequent use is required within a short period of time, it can be stored at 2-8℃.UsageThe following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.1.PCR reaction systemreagents50µl reaction systemfinal concentration2×Fast Probe Mixture25 µl1×Forward Primer, 10µM1µl0.2µM¹⁾Reverse Primer, 10µM1µl0.2µM¹⁾Probe, 10 µM1µl0.2µM²⁾Template DNA2µl³⁾ 50x Low ROX or High ROX(optional)⁴⁾1µl1×ddH₂Oup to 50µlNote: 1) Usually the primer concentration of 0.2µM can get better results, and 0.1-1.0µM can be used as a reference for setting the range. 2) The final concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, so please refer to the instruction manual of the instrument or the specific requirements of the use of each fluorescent probe for the adjustment of the concentration in actual use.(3) Usually the amount of DNA template is 10-100ng genomic DNA or 1-10ng cDNA as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be subjected to gradient dilution to determine the optimal amount of template to be used.(4) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.2. PCR reaction program:A two-step PCR reaction program is recommended, and this program is set up using the ABI 7500 Fluorescent Quantitative PCR Instrument as a reference.Note: 1) The enzyme used in this product must be pre-denatured at 95°C for 30s to achieve enzyme activation. Under this condition, most of the templates can be well unchained. For templates with high GC content and complex secondary structure, the pre-denaturation time can be extended to 1-4 minutes to allow the starting template to fully unchain.(2) It is recommended to use two-step PCR reaction program, if you do not get good experimental results due to the use of primers with lower Tm values, etc., you can try to carry out three-step PCR amplification, and the annealing temperature, please use the range of 56 ℃ - 64 ℃ as a setting reference... Read More | Mammalian lactate dehydrogenases (LDH) exist as five tetrameric isozymes composed of combinations of two different subunits. The H subunit predominates in heart muscle, which is geared for aerobic oxidation of pyruvate. The M subunit predominates in skeletal muscle and is concerned more with Mammalian lactate dehydrogenases (LDH) exist as five tetrameric isozymes composed of combinations of two different subunits. The H subunit predominates in heart muscle, which is geared for aerobic oxidation of pyruvate. The M subunit predominates in skeletal muscle and is concerned more with anaerobic metabolism and pyruvate reduction.Catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD+Recombinant rabbit muscle Lactate Dehydrogenase produced in E.Coli. Chromatographically purified. A lyophilized powder... Read More |