| Description | Catalase (CAT, EC 1.11.1.6) is widely found in animals, plants, microorganisms, and cultured cells. It is the primary H₂O₂-scavenging enzyme and plays a crucial role in the reactive oxygen species (ROS) scavenging system. H₂O₂ has a characteristic absorption peak at 240 nm. Catalase (CAT, EC 1.11.1.6) is widely found in animals, plants, microorganisms, and cultured cells. It is the primary H₂O₂-scavenging enzyme and plays a crucial role in the reactive oxygen species (ROS) scavenging system. H₂O₂ has a characteristic absorption peak at 240 nm. CAT decomposes H₂O₂, causing the absorbance of the reaction solution at 240 nm to decrease over time. The CAT activity can be calculated based on the rate of change in absorbance.Component50TStorageExtraction Buffer60 mL2-8℃Working Solution60 mL2-8℃User-Prepared Instruments and ReagentsUV spectrophotometer, benchtop centrifuge, adjustable pipettes, 1 mL quartz cuvette, mortar, ice, and distilled water.Experimental Procedure1. Crude Enzyme Extract Preparation1.1 Bacterial/Cell SamplesCollect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Use a bacteria/cell count (10⁴) to Extraction Buffer volume (mL) ratio between 500:1 and 1000:1 (recommended: 5 million bacteria/cells in 1 mL Extraction Buffer). Disrupt the bacteria or cells by sonication (ice bath, power 20% or 200W, pulse 3s on, 10s off, repeat 30 times). Centrifuge at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.1.2 Tissue SamplesUse a tissue mass (g) to Extraction Buffer volume (mL) ratio between 1:5 and 1:10 (recommended: weigh approx. 0.1 g tissue, add 1 mL Extraction Buffer). Homogenize in an ice bath. Centrifuge at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.1.3 Serum (Plasma) SamplesAssay directly.2. Assay Steps2.1 Preheat the spectrophotometer for at least 30 minutes. Set the wavelength to 240 nm. Zero the instrument with distilled water.2.2 Before assay, incubate the CAT Working Solution in a water bath at 37°C (for mammals) or 25°C (for other species) for 10 minutes.2.3 Add 35 µL of sample and 1 mL of Working Solution into a 1 mL quartz cuvette. Mix well and immediately measure the initial absorbance at 240 nm (A₁). Measure the absorbance again after 1 minute (A₂). Calculate ΔA = A₁ - A₂.3. CAT Activity Calculation3.1 Calculation of CAT Activity in Serum (Plasma)Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the degradation of 1 nmol of H₂O₂ per minute per milliliter of serum (plasma).Derived Formula:CAT Activity (nmol/min/mL) = [ΔA × Vtotal reaction÷ (ε × d) × 10⁹] ÷ Vsample÷ TSimplified Formula:CAT Activity (nmol/min/mL) = 678 × ΔA3.2 Calculation of CAT Activity in Tissues, Bacteria, or Cells(1) Based on Sample Protein ConcentrationUnit Definition: One unit of enzyme activity is defined as the amount that catalyzes the degradation of 1 nmol of H₂O₂ per minute per milligram of tissue protein.Derived Formula:CAT Activity (nmol/min/mg prot) = [ΔA × Vtotal reaction÷ (ε × d) × 10⁹] ÷ (Vsample× Cpr) ÷ TSimplified Formula:CAT Activity (nmol/min/mg prot) = 678 × ΔA ÷ Cpr(2) Based on Sample Fresh WeightUnit Definition: One unit of enzyme activity is defined as the amount that catalyzes the degradation of 1 nmol of H₂O₂ per minute per gram of tissue.Derived Formula:CAT Activity (nmol/min/g fresh weight) = [ΔA × Vtotal reaction÷ (ε × d) × 10⁹] ÷ (W × Vsample÷ Vtotal sample) ÷ TSimplified Formula:CAT Activity (nmol/min/g fresh weight) = 678 × ΔA ÷ W(3) Based on Bacterial or Cell DensityUnit Definition: One unit of enzyme activity is defined as the amount that catalyzes the degradation of 1 nmol of H₂O₂ per minute per 10⁴ bacteria or cells.Derived Formula:CAT Activity (nmol/min/10⁴ cells) = [ΔA × Vtotal reaction÷ (ε × d) × 10⁹] ÷ (500 × Vsample÷ Vtotal sample) ÷ TSimplified Formula:CAT Activity (nmol/min/10⁴ cells) = 1.356 × ΔAParameter Definitions:1.ΔA: Change in absorbance (A₁ - A₂)2.Vtotal reaction: Total reaction volume (1.035 × 10⁻³ L)3.ε: Molar extinction coefficient of H₂O₂ (4.36 × 10⁴ L/mol/cm)4.d: Light path length of the cuvette (1 cm)5.10⁹: Unit conversion factor (1 mole = 10⁹ nmoles)6.Vsample: Volume of sample added to the reaction (0.035 mL)7.T: Reaction time (1 min)8.Cpr: Sample protein concentration (mg/mL)9.W: Sample weight (g)10.Vtotal sample: Total volume of extraction buffer added (1 mL)11.500: Total number of bacteria or cells (5 million)Precautions1.Before formal testing, it is essential to perform a preliminary test with 2-3 samples expected to have significant differences.2.This product is for research use only. Not for use in clinical diagnosis.Frequently Asked Questions (FAQ)Q: What should I do if I get a negative value?A: Check if bubbles formed during the reaction. Excessive bubbling indicates very high enzyme activity, and bubbles can interfere, causing negative values. Try diluting the sample 10-fold with Extraction Buffer and re-assaying. If no bubbles form (with diluted sample or original reaction) and a small negative value persists, it indicates that the enzyme activity in this sample is below the detection limit... Read More | Product content:ComponentG665836100 rxnsG665836100 rxnsG665836100 rxns2×GoldStar Probe One Step Buffer1.4 ml1.4 ml1.4 mlGoldStar Probe One Step EnzymeMix100 µl100 µl100 µl50×Low ROX-50 µl-50×High ROX--50 µlRNase-Free Water1.5 ml1.5 ml1.5 mlProduct IntroductionProduct content:ComponentG665836100 rxnsG665836100 rxnsG665836100 rxns2×GoldStar Probe One Step Buffer1.4 ml1.4 ml1.4 mlGoldStar Probe One Step EnzymeMix100 µl100 µl100 µl50×Low ROX-50 µl-50×High ROX--50 µlRNase-Free Water1.5 ml1.5 ml1.5 mlProduct Introduction：This product is a specialized reagent kit for one-step Real Time RTqPCR using probe methods (TaqMan, Molecular Beacon, etc.). When using this product for Real Time RT qPCR reaction, reverse transcription and quantitative PCR are requiredConducted in the same reaction system, there is no need to add reagents or open the tube cap during the reaction process, avoiding contaminationThis has improved the efficiency of the experiment. This product has high detection sensitivity, strong fluorescence signal, and high signal-to-noise ratio, making it very suitable forDetection of RNA viruses and other trace amounts of RNA. The special buffering system it contains can enable reverse transcriptase to interact with DNA polymeraseMaximize the effectiveness and improve reaction efficiency. By using this product, a wider linear range can be obtained, which is beneficial for the target base Due to more accurate quantification, good repeatability, and high reliability.ROX dye is used to correct the fluorescence signal error generated between wells in quantitative PCR instruments, and is generally used for ABIReal Time PCR amplification equipment from companies such as Stratagene. The excitation optical systems of different instruments vary, thereforeThe concentration of ROX dye must be matched with the corresponding fluorescence quantitative PCR instrument.matters needing attention：1. Before using the reagents in this reagent kit, please gently mix them upside down to avoid foaming as much as possible, and use them after brief centrifugation. 2. This product uses RNA as a template for one-step RT-PCR experiments, and RNase contamination should be avoided during the operation process,2.It is recommended to perform RNA operations in a dedicated area, using specialized instruments and consumables. Operators should wear masks and disposable gloves and frequently change gloves. Experimental consumables should be treated with a 0.1% DEPC (diethyl pyrocarbonate) aqueous solution at 37 ℃ for 12 hours and sterilized under high pressure for 30 minutes before use.3. Each reagent in this kit should avoid repeated freezing and thawing as much as possible, as repeated freezing and thawing may lead to a decrease in product performance.4. This reagent kit must use specific primers, and the selection of primers can be based on specific experiments. The quality of primer design directly affects the results of RT qPCR reaction. When designing primers, GC content, primer length, and primer should be considered Due to factors such as location, secondary structure of PCR products, it is recommended to use professional primer design software for design.5. It is recommended to use specific probes in this reagent kit and use professional design software for design. Usage: The following examples are typical reaction systems and conditions. In practical operation, corresponding improvements and optimizations should be made based on the differences in template, primer structure, and target fragment size. (Please prepare the reaction solution on ice)1. Dissolve the RNA template, primers, 2xGoldStar Probe One Step Buffer, GoldStar Probe One Step EnzymeMix, and RNase Free Water and place them on ice for later use.2. PCR reaction system: reagent 25 µl Reaction system final concentration 2×GoldStar Probe One Step Buffer 12.5 µl 1× Forward Primer，10 µM 0.5 µl 0.2 µM 1） Reverse Primer，10 µM 0.5 µl 0.2 µM 1） Probe ，10 µM 0.5 µl 0.2 µM 2） GoldStar Probe One Step EnzymeMix 1.0 µl / RNA Template X µl 10 pg – 100 ng3） 50×Low ROX or High ROX （optional）4） 0.5 µl 1× RNase-Free Water up to 25 µl /Note: 1) Typically, the primer concentration is 0.2 µ M can achieve good results, ranging from 0.1 to 1.0 µ M serves as a reference for setting the range. 2) 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. Please refer to the instrument manual or the specific usage requirements of each fluorescent probe for concentration adjustment during actual use.3) The amount of RNA templates is usually based on 10 pg-100 ng as a reference. Due to the different copy numbers of target genes contained in templates of different species, gradient dilution can be applied to the templates to determine the optimal template usage.4) The excitation optical systems of different instruments vary, and depending on the instrument used for fluorescence quantification, 50 x Low ROX or 50 x High ROX can be added.3. Mix well, centrifuge briefly, and collect the solution to the bottom of the tube.4. RT-PCR reaction conditions steps temperature time / Reverse Transcription 45℃ 10 min / PCR pre denaturation 95℃ 10 min / denaturation 95℃ 15s 30-40cycle Annealing/Extension 60℃ 45s 30-40cycleAttention:1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 5-10 minutes.2) It is recommended to use a two-step PCR reaction program. If good experimental results cannot be obtained due to the use of primers with lower Tm values, a three-step PCR amplification can be attempted. The annealing temperature should be set within the range of 56 ℃ -64 ℃ as a reference... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Product contentS665868Component50 TStorageS665868ABuffer GL25 mLRTS665868BBuffer GW1 (concentrate)13 mLRTS665868CBuffer GW2 (concentrate)15 mLRTS665868DBuffer GE15 mLRTS665868EProteinase K2×1.25 mLRTS665868FSpin Columns DM with Collection Tubes50 setsRTProduct IntroductionThis kit is suitable Product contentS665868Component50 TStorageS665868ABuffer GL25 mLRTS665868BBuffer GW1 (concentrate)13 mLRTS665868CBuffer GW2 (concentrate)15 mLRTS665868DBuffer GE15 mLRTS665868EProteinase K2×1.25 mLRTS665868FSpin Columns DM with Collection Tubes50 setsRTProduct IntroductionThis kit is suitable for the extraction of genomic DNA from fresh saliva or saliva/preservation solution mixture.The purification process of this product does not require the use of toxic solvents such as phenol or chloroform, and ethanol precipitation is not necessary. The optimized buffer system enables DNA to bind heterogeneously to the silica matrix centrifugal adsorption column, and the inhibitors of PCR and other enzymatic reactions can be effectively removed by a two-step washing step, and finally eluted with a low-salt buffer or water to obtain high-purity DNA.The purified obtained can be directly used for enzyme digestion, PCR, Real-Time PCR, library construction, Southern Blot, molecular labeling and other downstream experiments.Self-contained reagent: anhydrous ethanolPre-experiment Preparation and Important Notes1. Repeated freezing and thawing of the sample should be avoided, as this may result in smaller fragments of extracted DNA and a decrease in the amount extracted.2. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the label of the reagent bottle before first use.3. Before use, please check whether Buffer GL appears to be crystallized or precipitated.Redissolve in a 56°C water bath.4. If the downstream experiments are sensitive to RNA contamination, 4 µL DNase-Free RNase A can be added in step 3(100 mg/mL).5. For prolonged storage of salivary DNA at room temperature, our Salivary DNA Preservation Tubes are recommended.Operation steps1. Add 400 µL of saliva sample or saliva/preservation solution mixture.Note: 1) Saliva mixtures added to the preservation solution require a 50°C water bath for 1 hour or an empty 50°C temperature chamber for 2 hours prior to extraction.2) If an increase in sample volume is required, multiply the volumes of Proteinase K, Buffer GL, and anhydrous ethanol in Steps 2-4, and the liquid can be transferred in multiple times in Step 5.2. Add 40 µL of Proteinase K.3. Add 400µL Buffer GL, vortex and shake to mix thoroughly, and water bath at 56℃ for 15-30 minutes.Note: If RNA removal is required, add 4 µL of RNase A solution at a concentration of 100 mg/mL after the above steps are completed, vortex for 15 seconds, and leave at room temperature for 2 minutes.4. Centrifuge briefly to remove water droplets from the inside of the tube cap. Add 400 µL of anhydrous ethanol and mix well by vortexing and shaking. Centrifuge briefly.Note: 1) Vortex and shake to mix immediately after adding Buffer GL and anhydrous ethanol.The addition of Buffer GL and anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.2) A sol-gel product may be formed after GL and anhydrous ethanol, in which case vigorous shaking or vortexing is recommended.3) The solution obtained in the previous step is added to the adsorption column in the Collection Tube.5. (Spin Column DM) in the collection tube, and if the solution cannot be added all at once, it can be transferred in several times. centrifuge at 12,000 rpm (∼13,400 × g) for 1 min, pour off the waste solution in the collection tube, and put the adsorption column back into the collection tube.6. Add 500 µL of Buffer GW1 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.7. Add 500 µL of Buffer GW2 to the adsorption column (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.Note: Step 7 can be repeated if further DNA purity is required.8. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Leave the adsorption column at room temperature for several minutes to dry thoroughly.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).9. Place the adsorption column in a new centrifuge tube (supplied), add 50-200 µL of Buffer GE or sterilized water to the middle of the adsorption column overhanging the column, let it stand at room temperature for 2-5 minutes, and centrifuge at 12,000 rpm for 1 minute to collect the DNA solution.-20°C to preserve DNA.Note: 1) If the downstream experiment is sensitive to pH or EDTA, you can use sterilized water for elution. The pH of the eluent has a great influence on the elution efficiency, if water is used as the eluent should ensure that its pH is 7.0-8.5 (you can use NaOH to adjust the pH of the water to this range), and the elution efficiency is not high when the pH is lower than 7.0.2) Buffer GE preheated in a 65-70°C water bath and incubated at room temperature for 5 min before centrifugation can increase the yield.3) Because DNA preserved in water is subject to acidic hydrolysis, for long-term storage, elution with Buffer GE and storage at -20°C is recommended... Read More |