| Description | Chloroplast 3-phosphoglycerate kinase (PGK) is a key enzyme in the Calvin cycle. Detection Principle: Chloroplast 3-phosphoglycerate kinase catalyzes the reaction of 3-phosphoglyceric acid and ATP to produce 1,3-bisphosphoglyceric acid. The latter, under the action of glyceraldehyde-3-phosphate Chloroplast 3-phosphoglycerate kinase (PGK) is a key enzyme in the Calvin cycle. Detection Principle: Chloroplast 3-phosphoglycerate kinase catalyzes the reaction of 3-phosphoglyceric acid and ATP to produce 1,3-bisphosphoglyceric acid. The latter, under the action of glyceraldehyde-3-phosphate dehydrogenase and NADH, produces glyceraldehyde-3-phosphate and NAD⁺. The enzyme activity of 3-phosphoglycerate kinase is determined by measuring the decrease in NADH.Component96TStorageExtraction Buffer 1100 mL2-8℃Extraction Buffer 2100 mL2-8℃Reagent 11EA-20℃. Store in the dark.Reagent 23EA2-8℃Reagent 31EA-20℃Reagent 435 mL2-8℃Reagent 51EA-20℃Reagent Preparation:Reagent 1 (Powder, 1 vial):Before opening, ensure the powder is at the bottom (can be flicked manually).Add 1.1 mL of distilled water to dissolve. Use after preparation.The prepared solution can be stored for the duration of the kit's validity period.Reagent 2 (Powder, 3 vials):Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom.Add 0.4 mL of distilled water per vial to dissolve. Use after preparation.Unused dissolved reagent can be aliquoted and stored at -20°C. Avoid repeated freeze-thaw cycles. Use within 3 days.Reagent 3 (Powder, 1 vial):Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom.Add 1.1 mL of distilled water to dissolve. Use after preparation.The prepared solution can be stored for the duration of the kit's validity period.Reagent 5 (Powder, 1 vial):Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom.Add 1.1 mL of distilled water to dissolve. Use after preparation.The prepared solution can be stored for the duration of the kit's validity period.User-Prepared Instruments and MaterialsMortar (Homogenizer), Ice box (Ice maker), Benchtop centrifuge, Adjustable micropipettes, Water bath (Oven, Incubator, Metal bath), 96-well plate, Centrifuge tubes, Microplate reader, Vortex mixer/shaker, Distilled water (Deionized water or Ultrapure water are acceptable).Experimental ProcedureIt is recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure and to determine or adjust sample concentrations based on the preliminary results, preventing unnecessary waste of samples or reagents.1. Sample Extraction (Chloroplast Isolation)Weigh approximately 0.1 g of plant tissue sample. Add 1 mL of Extraction Buffer 1 and homogenize rapidly in an ice bath. Centrifuge at 1,600 rpm, 4°C for 5 minutes. Discard the pellet. Take the supernatant and centrifuge again at 5,000 rpm, 4°C for 15 minutes. Discard the supernatant and keep the pellet. Add 1 mL of Extraction Buffer 2 to the pellet. Vortex vigorously for 15 seconds. Place on ice (or in a refrigerator) and incubate at 4°C for 15 minutes. Centrifuge at 13,000 rpm, 4°C for 5 minutes. Collect the supernatant for assaying the chloroplast 3-phosphoglycerate kinase (PGK) enzyme activity.Important: The entire chloroplast extraction process must be maintained at 4°C.Note: If increasing the sample amount, maintain a tissue mass (g) to Extraction Buffer volume (mL) ratio between 1:5 and 1:10.2. Assay Steps2.1 Preheat the microplate reader for 30 minutes. Set the wavelength to 340 nm and the temperature to 25°C.2.2 Thaw all reagents to room temperature (25°C).2.3 Add reagents sequentially to a 96-well plate:ReagentTest Well (µL)Sample20Reagent 110Reagent 210Reagent 310Reagent 4140Mix well and incubate at room temperature (25°C) for 10 minutes.Reagent 510Mix gently. Under room temperature (25°C) conditions, read the absorbance at 340 nm at 30 seconds (A₁) and then again after 10 minutes (A₂). Calculate ΔA = A₁ - A₂.注：Notes:(1) If ΔA is close to zero, the reaction time can be appropriately extended to 20 minutes before reading A₂. If the reaction time is changed, the new time (T) must be substituted into the calculation formula. Alternatively, the sample volume can be increased (e.g., to 40 µL, with a corresponding decrease in Reagent 4 volume); the new sample volume (V₁) must then be substituted into the calculation formula.(2) If the decrease trend is unstable, read the absorbance every 20 seconds and select a linearly decreasing time period for calculation. The corresponding A values for this period should be used to calculate ΔA and substituted into the formula.(3) If the initial absorbance A₁ is too high (e.g., >2, as in dark green plant leaves with high pigment content), consider appropriately reducing the sample volume; the new sample volume (V₁) must be substituted into the calculation formula. Alternatively, add a small amount of activated carbon to the sample, mix, let stand for 5 minutes, then centrifuge at 12,000 rpm, 4°C for 10 minutes, and use the supernatant for assay.(4) If ΔA is greater than 0.5, reduce the reaction time (e.g., to 5 minutes) or reduce the sample volume (e.g., to 10 µL). The changed reaction time (T) and/or sample volume (V₁) must be substituted into the calculation formula.3. Calculation of Results3.1 Based on Sample MassUnit Definition: One unit of enzyme activity is defined as the amount that consumes 1 nmol of NADH per minute per gram of tissue.Derived Formula: chl PGK (nmol/min/g fresh weight) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (W × V₁ ÷ V) ÷ TSimplified Formula: chl PGK (nmol/min/g fresh weight) = 321.6 × ΔA ÷ W3.2 Based on Sample Protein ConcentrationUnit Definition: One unit of enzyme activity is defined as the amount that oxidizes 1 nmol of NADH per minute per mg of tissue protein.Derived Formula: chl PGK (nmol/min/mg prot) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (V₁ × Cpr) ÷ TSimplified Formula: chl PGK (nmol/min/mg prot) = 321.6 × ΔA ÷ CprParameter Definitions:ε: Molar extinction coefficient of NADH (6.22 × 10³ L/mol/cm)d: Light path length for the 96-well plate (0.5 cm)V: Volume of Extraction Buffer added to the pellet (1 mL)V₁: Volume of sample added to the reaction (0.02 mL)V₂: Total volume of the reaction system (0.2 mL = 2.0 × 10⁻⁴ L)T: Reaction time (10 minutes)W: Sample weight (g)Cpr: Sample protein concentration (mg/mL); Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.Precautions It is strongly recommended to first perform a preliminary test using 1-3 samples with expected significant differences (e.g., different types or groups) to familiarize yourself with the procedure. Based on the preliminary results, determine or adjust sample concentrations to prevent unnecessary waste of samples or reagents... Read More | Product IntroductionAlamar Blue detection reagent provides a simple, rapid, reliable and safe method for cell proliferation and cytotoxicity detection, which is suitable for high-throughput detection experiments. The main component of the detection reagent is a redox indicator. In the oxidized stateProduct IntroductionAlamar Blue detection reagent provides a simple, rapid, reliable and safe method for cell proliferation and cytotoxicity detection, which is suitable for high-throughput detection experiments. The main component of the detection reagent is a redox indicator. In the oxidized state, it appears purple-blue and non-fluorescent, while in the reduced state, it turns into a reduction product with pink or red fluorescence, with an absorption peak of 530-560nm and an emission peak of 590nm.In the process of cell proliferation, the ratios of NADPH/NADP, FADH/FAD, FMNH/FMN and NADH/NAD in the cell increase and are in a reducing environment. The dye taken into the cell is reduced by these metabolic intermediates and cytochromes and then released outside the cell and dissolved in the culture medium, changing the culture medium from non-fluorescent indigo blue to fluorescent pink. Finally, use an ordinary spectrophotometer or fluorophotometer for detection, and the absorbance and fluorescence intensity are proportional to the number of active cells.Instructions1. Add 10µl of detection reagent to 100µl of cell suspension, and incubate in a cell incubator for 2-6 hours. The color of the medium changes from indigo blue to pink and you can proceed to the next step.2. It is recommended to use a fluorescence microplate reader for detection, the excitation light wavelength is between 530-560 nm, the emission light wavelength is 590 nm, and the relative fluorescence unit (RFU) is recorded.3. Draw a standard curve or cell growth curve: the ordinate (Y axis) is the relative fluorescence unit (RFU); the abscissa (X axis) is the cell number or time point or drug concentration.Precautions1. The appropriate density of cells can increase the detection sensitivity. For 96-well plates, we recommend seeding 100 microliters of cells per well. The cell concentration range is: 100-10,000/well for adherent cells, 2,000-50,000/well for suspension cells, and medium as a blank control. For 384-well plates, the cell concentration and seeding volume are both halved.2. The whole process should be aseptic operation, because microbial contaminants can also reduce the detection reagents and affect the experimental results.3. Pay attention to the concentration of inoculated cells and the incubation time after adding detection reagents. If the cell concentration is too high or the incubation time is too long, it will cause a secondary reduction reaction, resulting in colorlessness and disappearance of fluorescence.4. When incubating, avoid light.5. This product can use fluorescence or spectrophotometric detection, but the sensitivity of fluorescence is high, and the experimental error is small. Fluorescence detection is recommended... Read More | Inquire | Product DescriptionOur Glycan Sequencing Kit includes the enzymes and buffer required to sequence ten N-linked oligosaccharides.ContentsNeuraminidase from Arthrobacter ureafaciens – 80 µlBeta-Galactosidase from Streptococcus pneumoniae – 60 µlN-Acetylglucosaminidase from Product DescriptionOur Glycan Sequencing Kit includes the enzymes and buffer required to sequence ten N-linked oligosaccharides.ContentsNeuraminidase from Arthrobacter ureafaciens – 80 µlBeta-Galactosidase from Streptococcus pneumoniae – 60 µlN-Acetylglucosaminidase from Streptococcus pneumoniae) – 40 µlAlpha-Mannosidase from Jack Bean – 20 µlCore Alpha-Mannosidase from X. manihotis) – 10 µl5X Reaction buffer – 400 µlAnalysisMany methods of analysis are available, including HPLC, gel electrophoresis, HPAEC, capillary electrophoresis, and mass spectrometry. For more information on these methods, please contact us.StabilityThe Glycan Sequencing Kit is stable at least 12 months when stored properly. Several days exposure to ambient temperatures will not reduce activity.PurityAll Enzymes are tested for contaminating protease by incubating 10 µg of denatured BSA with 2 µl of enzyme at 37°C for 24 hours. SDS-PAGE analysis of the treated BSA shows no evidence of degradation.The production host strains for our recombinant enzymes have been extensively tested and do not produce any detectable glycosidases. Enzymes purified from native sources are tested for contaminating exoglycosidases The absence of exoglycosidase contaminants is confirmed by extended incubations with the corresponding pNP-glycosides... Read More | Inquire |