| Description | GoldStar DNA Polymerase is a chemically modified and highly efficient Taq DNA Polymerase. The enzyme's activity is completely blocked at room temperature, making it inactive at low or room temperature. This effectively avoids non-specific amplification caused by non-specific binding of primers and GoldStar DNA Polymerase is a chemically modified and highly efficient Taq DNA Polymerase. The enzyme's activity is completely blocked at room temperature, making it inactive at low or room temperature. This effectively avoids non-specific amplification caused by non-specific binding of primers and templates or primer dimers at room temperature. Enzyme activation requires incubation at 95 ℃ for 10 minutes. The unique buffer system makes the application of enzymes more extensive, enabling efficient amplification of high GC content, complex secondary structures, and low copy templates. Using this product for PCR amplification, the 3 'end of the PCR product contains an "A" base, which can be directly used for T/A cloning. This product has strong specificity and does not require gel recovery to remove impurities after PCR amplification. It can be directly used for downstream cloning or chip hybridization experiments. Mainly used for routine PCR, RT-PCR, Real time PCR, multiplex PCR, gene chip analysis, and SNP detection, especially suitable for PCR reactions that require high specificity.G665840Component500 U2500 UStorageG665840AGoldStar DNA Polymerase, 5 U/µL100 µL 5×100 µL-20℃. Avoid freeze/thaw cycle.G665840B5×GoldStar PCR Buffer1.9 mL 5×1.9 mL-20℃. Avoid freeze/thaw cycle. Attention: The 5 x GoldStar Taq PCR Buffer of this product contains 8.5mM magnesium ions.Unit DefinitionUsing activated salmon sperm DNA as a template/primer, the amount of enzyme required to incorporate 10 nmol of deoxyribonucleotide into acidic insoluble substances is defined as 1 active unit (U) at 74 ℃ for 30 minutes.Quality controlSDS-PAGE detection shows a purity greater than 99%; No exogenous nuclease activity detected; PCR method for detecting host free residual DNA; Can effectively amplify single copy genes in the human genome. Store at room temperature for one month without significant changes in activity.UsageThe following is an example of a PCR reaction system and reaction conditions for amplifying a 1 kb fragment using human genomic DNA as a template. In practical operation, corresponding improvements and optimizations should be made based on the template, primer structure, and target fragment size.1. PCR reaction system Reagent 50 µl Reaction system Final concentration 5×GoldStar PCR Buffer 10 µl 1 × dNTP Mix，10 mM each 1 µl 200 µM each Forward Primer，10 µM 2 µl 0.4 µM Reverse Primer，10 µM 2 µl 0.4 µM Template DNA <0.5 µg <0.5 µg/50 µl GoldStar DNA Polymerase，5 U/µl 0.5 µl 2.5 U/50 µl ddH2O up to 50 µl / Attention: Primer concentration, please use final concentration of 0.1-1.0 µ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the primer concentration can be reduced to optimize the reaction system.2. PCR reaction conditions Step Temperature Time / Pre denaturation 95℃ 10 min / Denaturation 95℃ 30 s 30-40 cycles Annealing 55-65℃ 30 s 30-40 cycles Extension 72℃ 60 s 30-40 cycles Final extension 72℃ 5 min / Attention:1) In general experiments, the annealing temperature is 5 ℃ lower than the melting temperature Tm of the amplification primer, and the annealing time is generally 30-60 seconds. If the ideal amplification efficiency cannot be achieved, the annealing temperature should be appropriately reduced; When non-specific reactions occur, increase the annealing temperature to optimize the reaction conditions.2) The extension time should be set according to the size of the amplified fragment. The amplification efficiency of GoldStar Taq DNA Polymerase contained in this product is 1-2 kb/min.3) The number of cycles can be set based on the downstream application of the amplification product. If the number of cycles is too small, the amplification amount is insufficient; If there are too many cycles, the probability of mismatches will increase, and non-specific backgrounds will be severe. So, while ensuring product yield, the number of cycles should be minimized as much as possible.4) This product must achieve enzyme activation under pre denaturation conditions of 95 ℃ and 10 minutes... Read More | General DescriptionNatural human C5a is prepared from human C5 protein cleaved into C5a and C5b by human C5 convertase. The C5a is converted to C5a desArg by proteolytic removal of the C-terminal arginine. The primary carboxypeptidase responsible for Arg removal is serum carboxypeptidase N, but General DescriptionNatural human C5a is prepared from human C5 protein cleaved into C5a and C5b by human C5 convertase. The C5a is converted to C5a desArg by proteolytic removal of the C-terminal arginine. The primary carboxypeptidase responsible for Arg removal is serum carboxypeptidase N, but there are several different carboxypepticases in serum. C5a desArg is a naturally glycosylated polypeptide containing 73 amino acids with a molecular weight of approx. 10,250 daltons. It contains 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). C5a desArg is produced when C5a is“inactivated” by removal of its C-terminal arginine amino acid. This cleavage occurs by the action of the plasma enzyme carboxypeptidase N. 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. Its biological properties include being weakly chemotactic for neutrophils (PMN), causing smooth muscle contraction, increasing vascular permeability, causing histamine and TNF-alpha release, and causing lysosomal degranulation of immune cells. C5a and C5a desArg act 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 nonimmunolgical functions in organ development, CNS development, neurodegeneration, tissue regeneration and hematopoiesis (Monk, P.N. et al. (2007)).Native versus Recombinant C5a desArgNumerous recombinant forms of C5a and C5a desArg are sold by many companies. In side-by-side biological testing, we have found that our native native proteins are 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 & StructureDeglycosylated MW: Calculated monoisotopic mass 8112; Calculated average mass 8117.Isoelectric point: pI = 8.8Carbohydrate content: ~25% carbohydrate (heterogeneous) Amino acid sequence: TLQKKIEEIA AKYKHSVVKK CCYDGACVNN DETCEQRAAR ISLGPRCIKA FTECCVVASQ LRANISHKDM QLGMDL Number: MFCD00130842NMRderived structure: FEBS Lett. 238:289-294, 1988; Biochemistry 28:172-185,1989; Biochemistry 29:2895-2905, 1990; Proteins 28:261-267, 1997.Extinction Coeff. A280 nm = 0.41 at 1.0 mg/mlPurity: > 97% by SDS-PAGEAssaysThe multitude of biological functions of C5a has resulted in the use of many different assay systems. 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. Functional responses have been detected in the picomolar concentration range (Gerard, C. et al. (1981); Hugli, T.E. et al. (1981)).ELISA kits for the assay of C5a and C5a desArg in blood and other fluids are sold by many companies. 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 of C5a desArg has been reported to be approximately 4 nM although it is difficult to draw, store and test 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 (sub-picomolar C5a).RegulationC5adesArg levels are regulated by two processes: formation 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 desArg is created when C5a is“inactivated” by removal of its C-terminal arginine amino acid. The product C5a desArg is produced by the action of the plasma enzyme carboxypeptidase N. 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 and C5a desArg results in their rapid removal from circulation.DeficienciesA deficiency of C5 or a deficiency of the enzymes that cleave C5 to generate C5a would result in the absence of C5a and C5a desArg. A knock-out mouse deficient in carboxypeptidase N has been created and found to be hypersensitive to complement activation and CVF administration (Mueller-Ortiz S.L. et al. (2009)). Administration of human C5a was 100% lethal in these KO mice probably due to their inability to inactivate C5a to C5a desArg. 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 and C5a desArg. Humans lacking C5 are susceptible to repeated infections from a wide variety of organisms, primarily gram-negative 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 due to the absence of C5a and C5a desArg is unclear but information on this isbeing 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 3MSDS available upon request... Read More | Human PTHrP-(1-36) is a secretory form of PTHrP with anticalciuric effects. Human PTHrP-(1-36) enhances beta cell function and proliferation. Human PTHrP-(1-36) can be used in the research of humoral hypercalcemia of malignancy (HHM) and hyperparathyroidism.In VitroHuman PTHrP-(1-36) (EC 50 : 0.05 Human PTHrP-(1-36) is a secretory form of PTHrP with anticalciuric effects. Human PTHrP-(1-36) enhances beta cell function and proliferation. Human PTHrP-(1-36) can be used in the research of humoral hypercalcemia of malignancy (HHM) and hyperparathyroidism.In VitroHuman PTHrP-(1-36) (EC 50 : 0.05 nM) increases intracellular calcium in human epidermal keratinocytes. Human PTHrP-(1-36) (100 nM, 24 h) increases human β-cell proliferation. Human PTHrP-(1-36) (100 nM, 30 min) enhances insulin secretion in human islets. PTHrP-(1-36) (mouse, EC 50 : 1 nM) induces a rapid Ca 2+ response in UMR 106 cells. MCE has not independently confirmed the accuracy of these methods. They are for reference only.In VivoPTHrP-(1-36) (mouse, 160 µg/kg, s.c., for 5 days/week for 7, 30, or 90 days) enhances beta cell regeneration and increases beta cell mass in a mouse model of partial pancreatectomy. PTHrP-(1-36) (mouse, 100 µg/kg, s.c., every other day) reverses the observed decrease of Wisp1 expression in the diabetic mice. MCE has not independently confirmed the accuracy of these methods. They are for reference only.Form:Solid... Read More | Purity: >95%, by SDS-PAGE visualized with Coomassie® Blue Staining. Description： CD4, also known as L3T4, T4, and W3/25, is an approximately 55 kDa type I transmembrane glycoprotein that is expressed predominantly on thymocytes and a subset of mature T lymphocytes. It is a standard Purity: >95%, by SDS-PAGE visualized with Coomassie® Blue Staining. Description： CD4, also known as L3T4, T4, and W3/25, is an approximately 55 kDa type I transmembrane glycoprotein that is expressed predominantly on thymocytes and a subset of mature T lymphocytes. It is a standard phenotype marker for the identification of T cell populations. Mature human CD4 consists of a 371 amino acid (aa) extracellular region containing four immunoglobulin-like domains, a 22 aa transmembrane segment, and a 40 aa cytoplasmic domain. Within the ECD, human CD4 shares approximately 52% aa sequence identity with mouse and rat CD4. CD4 is expressed along with CD8 on double positive T cells during their development in the thymus. Either CD4 or CD8 expression is then lost, giving rise to single positive (SP) CD4+ or CD8+ mature T cells. CD4+ SP cells, also known as T helper cells, further differentiate into multiple subsets of CD4+ cells including Th1, Th2, Th17, Tfh, and Treg cells which regulate humoral and cellular immunity. CD4 is reexpressed on circulating CD8+ T cells upon activation and contributes to their cytotoxic effector activity. In human, CD4 is additionally expressed on macrophages, neutrophils, monocytes, NK cells, and neurons and glial cells in the brain. Similar CD4 distribution between species cannot be assumed as demonstrated by its presence on macrophages in human and rat but not in mouse. CD4 binds directly to MHC class II molecules on antigen presenting cells. This interaction contributes to the formation of the immunological synapse which is focused around the TCR-MHC class II-antigenic peptide interaction. Palmitoylation of two cysteine residues in the cytoplasmic tail of CD4 promotes the localization of CD4 in lipid rafts and its ability to augment TCR signaling via activation of the tyrosine kinase Lck. CD4 also functions as a chemotactic receptor for IL-16 and, in human, as a co-receptor for the gp120 surface glycoprotein of HIV-1... Read More | TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for measuring HRP probe activity. A stable blue precipitate is formed at the reaction site.The substrate does not contain NMP (1-methyl2-pyrrolidone) making it REACH Restricted Substances List Annex XVII compliant, while ensuring maximal safety during use, and minimal negative environmental impact.Product Characteristics TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for measuring HRP probe activity. A stable blue precipitate is formed at the reaction site. The substrate does not contain NMP (1-methyl-2- pyrrolidone) making it REACH Restricted Substances List Annex XVII compliant, while ensuring maximal safety during use, and minimal waste problems after use.Composition & Properties Ready-to-use substrate: Includes substrate buffer and hydrogen peroxide. No other reagents should be added.Working Procedure The following procedure is applicable to nitrocellulose membranes. The procedure must be optimized for other membranes.1.The desired amount of substrate is poured into a sealed container and allowed to reach room temperature, in the dark, before use. 2.After the last incubation with HRP-labelled Streptavidin or HRP-labelled secondary antibody it is recommended to wash the membrane in a 0.1 M Tris buffer pH 7.4.3.Shake off the excess buffer and incubate the membrane in the TMB-D Blotting solution for 10 minutes. 4.Wash the membrane in distilled water and allow it to dry. 5.The site of positive reaction will appear light blue with no or very little background staining.Tips & Tricks • The membrane can be blocked with Kementec’s Synthetic Blocking Buffer for Blotting, (cat. no. S494457). • For long-term preservation of the results, the membranes must be stored in the dark.Handling & Storage • Store solution at 2-8⁰C in the dark. • Avoid exposure to light, heat and contamination with metal ions or peroxidase. • Re-dispense only into bottles made of High-Density Polyethylene (HDPE), amber color. Dispensing guidelines are available upon request... Read More |