| Description | Absorbance: A412 nm = 1.10 at 1/50 dilution in deionized waterGeneral DescriptionEr are rabbit red blood cells which have been washed free of rabbit plasmaproteins, but they are not coated with antibodies as is done with EA. These cells have traditionally been used as cells that spontaneously Absorbance: A412 nm = 1.10 at 1/50 dilution in deionized waterGeneral DescriptionEr are rabbit red blood cells which have been washed free of rabbit plasmaproteins, but they are not coated with antibodies as is done with EA. These cells have traditionally been used as cells that spontaneously activate the human alternative complement pathway in serum samples (Morgan, B.P. (2000; Dodds, A.W. and Sim, R.B. (1997)). Most human serum samples have small amounts of natural antibodies (usually IgG) to rabbit antigens and at high concentrations of serum these will agglutinate Er. This will activate the classical pathway if not blocked. Blocking the classical pathway is usually accomplished with 3 to 13 mM MgEGTA.Er are supplied at assay-ready concentrations in the traditional buffer used for APH50 assays (GVB°) which lacks metal ions. They can usually be used for 2 weeks after preparation. They are shipped cold, but are not harmed by extended periods at room temperature (note that they circulate 60+ days at 37℃in vivo). They should be washed once before each use (3-5 min at 500 to 1000 x g at 4℃) and resuspended in GVB° to reduce background and readjust their concentration. This procedure may also be used to concentrate the cells.Physical CharacteristicsEr are natural uncoated (non-opsonized) rabbit erythrocytes.AssaysAlthough the APH50 titer is widely used and serves as the primary test for alternative pathway complement activity, the APH50 assay procedure is not entirely standardized. In research labs there are as many procedures as there are labs, but all are basically similar and give useful results. The main difference between the classical pathway assays for CH50 determination and the alternative pathway assays for APH50 is the concentration of serum required. Alternative pathway activity becomes ineffective at dilutions of serum beyond 1/10 to 1/30. In contrast, CH50 titers are performed in the range 1/100 to 1/500 dilution. Dilution silences the alternative pathway in CH50 titers, but APH50 measurements would be overwhelmed by the classical pathway if this pathway were not blocked. Blocking of alternative pathway activity may be accomplished by using serum depleted of factor B or D or most conveniently by adding 3 to 13 mM MgEGTA (final concentration) to the assays which completely inhibits the classical pathway by chelating calcium ions and dissociating C1. Briefly, a complement-containing serum sample is diluted such that the final dilutions in the assay are in the range from 1/2 to 1/20. Controls include two tubes with no complement for the 0% lysis background and two tubes for 100% lysis. Assay tubes should be set up on ice. The assays are performed in 200 µL containing 4 to 60 µL of test serum, 10 µL 0.1 M MgEGTA, GVB°, and 50 µL Er. The assays are mixed and placed in a 37℃ water bath with remixing at approximately 10 min intervals. After 30 min, 2 mL of cold GVBE is added, mixed and the unlysed cells are spun down (500-1000 x g for 3 min). The absorbance of the supernatant should be determined at 412 nm in a 1 cm cuvette and the percentage of specific lysis calculated after subtracting the background and dividing by the 100% lysis control. The data is plotted as percent lysis on the y axis and µL serum on the x axis. The APH50 value of the complement sample is calculated from the amount of serum needed to cause lysis of 50% of the cells. For example, if it takes 12 µL NHS to lyse 50% of the cells then this serum would contain 83 AHP50 units/mL (1000 µL /12 µL = 83 units/mL). It should be noted that somewhat different APH50 values can be obtained on what should be identical samples. This is the result of the vast number of variables involved in APH50 determinations including the fact that each batch of rabbit erythrocytes is slightly different.ApplicationsEr cells are primarily used to determine the activity of the alternative pathway of complement (APH50 titer) (Morgan, B.P. (2000; Dodds, A.W. and Sim, R.B. (1997)). Natural antibodies present in human blood to animal antigens may cause agglutination of the cells. This antibody may also cause lysis if the classical pathway is not blocked.Rabbit erythrocytes are attacked and lysed by the alternative pathways of most mammalian species. It has not been clearly demonstrated why rabbit complement does not lyse rabbit Er when most other animal species do lyse these cells, although they do carry rabbit DAF which works on rabbit, but not human complement enzymes.RegulationRabbit erythrocytes (Er) are used for human complement assays partly for convenience, but also because they lack membrane-bound regulators of human complement. No significant level of functional DAF, CD59 or CR1 exists on Er for human complement. Thus, Er are useful for their lack of membrane regulatory activities... Read More | ProductsThis product is a high purity genomic DNA extract from 293T cells, agarose gel (0.7%) electrophoresis showed that the size of the DNA extract is more than 15Kb, and basically no degradation, the product is ultimately preserved in TE Buffer, which can be widely used in molecular biology ProductsThis product is a high purity genomic DNA extract from 293T cells, agarose gel (0.7%) electrophoresis showed that the size of the DNA extract is more than 15Kb, and basically no degradation, the product is ultimately preserved in TE Buffer, which can be widely used in molecular biology experiments, such as PCR, enzyme digestion, hybridization, microarray analysis, and other molecular biology experiments.The product was quantified using NanoDrop One at a concentration of 200 ng/µL.Preparation and precautions before useLong-term storage at -20˚C is recommended. Before use, the bottle should be removed from the refrigerator and equilibrated to room temperature and centrifuged before opening the cap for use. Samples should be restored to the sealed state as soon as possible after opening.How to use (take qPCR experiment as an example)1. Amplification template preparationThe samples to be detected were diluted with TE (10 mM Tris-Cl, pH 8.0,1 mM EDTA), and the concentration after dilution was as close as possible to the range of 0.05-10 ng/µL. The samples were placed on ice at 4°C and set aside.2. Standard dilution: according to the following table, firstly dilute Human DNA Standard 1 (100ng/uL) with TE to make 5 different concentrations of standards according to the table below. 10ng/µL of DNA Standard 1 (Std. 1) can be stored stably at -20℃ for 1 month; Std2-5 can only be used on the same day, and should be placed at 4℃ or on ice when not in use for the time being after preparation. When not used temporarily after preparation, it should be stored at 4℃ or on ice.styleCorresponding concentration (ng/µL)Minimum dilution volume (in µL)Std.11010 [100 ng/µL DNA Standard 1] + 90 TEStd.22.520 [Std. 1] +60 TEStd.30.62520 [Std. 2] +60 TEStd.40.1562520 [Std. 3] +60 TEStd.50.039062520 [Std. 4] +60 TE3. qPCR reaction system preparationThe cryopreserved reagents to be used were completely thawed and mixed by inversion several times before preparation, and then briefly centrifuged and prepared for use. 20 µL of the base reaction system was as follows.The base reaction system for 20 µL was as follows:reagents20µL reaction system2×qPCRMix10µLPrimerMixXµLProbeMixXµLTemplate4µLddH2OMake up to 20 µLNote: High Rox model: add 1 µL of 50×High Rox per 50 µL of reaction system; Low Rox model: add 1 µL of 50×High Rox per 500 µL of reaction system.Usually, better results can be obtained with a primer concentration of 0.2 µM, and 0.1-1.0 µM can be used as a reference for setting the range.The 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 manual of the instrument or the specific requirements for the use of each fluorescent probe for the adjustment of the concentration during actual use.Prepare a sufficient amount of reaction system mixture as required. After the reaction system has been prepared and mixed thoroughly, add 16 µL per well to the reaction wells. Then add the prepared standard and diluted sample into the corresponding reaction wells, the volume of addition is 4µL/well. TE was added to the blank control tube, and the same amount of TE was added at 4 µL/well.It is recommended to use 20 µL for the reaction, if you need to perform a smaller system reaction, reduce the system components in equal proportion.4. qPCR reaction programThe following is an example of our GoldStar Probe Mixture reaction conditions, which should be improved and optimized according to the PCR product template, primer structure and target fragment size.movetemptimingcirculatepremutability95°C10min1denaturation95°C10sec55Annealing/Extension60°C30sec5Data analysis1. Standard curve productionThe standard curve was plotted with reference to the Excel sheet for data processing. The correlation coefficient R2 of the standard curve should not be lower than 0.98, and the slope should be between -3.1 and -3.6 when the Ct value is the vertical coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment... Read More | Inquire | 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 | Tyrosine decarboxylase catalyzes the removal of the carboxyl group from tyrosine to produce tyramine and carbon dioxide. Pyridoxal 5'-phosphate is a necessary cofactor. By using the apoenzyme prepared from cells grown on a vitamin B6 deficient medium pyridoxal phosphate may be determined. The Tyrosine decarboxylase catalyzes the removal of the carboxyl group from tyrosine to produce tyramine and carbon dioxide. Pyridoxal 5'-phosphate is a necessary cofactor. By using the apoenzyme prepared from cells grown on a vitamin B6 deficient medium pyridoxal phosphate may be determined. The HOLOenzyme may be used to determine tyrosine, phenylalanine and dihydroxyphenylalanine either manometrically or colorimetrically.L-Tyrosine decarboxylase apoenzyme from Streptococcus faecalis has been used in a study to purify and characterize tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase.L-Tyrosine decarboxylase apoenzyme from Streptococcus faecalis has also been used in a study to investigate the stereospecificity of sodium borohydride reduction of tyrosine decarboxylase.One Unit yields 1µmole of CO2 per minute from L-tyrosine at 37°C, pH 5.5. The APOenzyme activity is measured in the presence of excess pyridoxal phosphate... Read More |