| Description | Pyruvate Kinase (PK) is widely present in animals, plants, microorganisms, and cultured cells. It catalyzes the final step of glycolysis, serving as one of the key rate-limiting enzymes in this process and a crucial enzyme for ATP production. Therefore, determining PK activity is of significant Pyruvate Kinase (PK) is widely present in animals, plants, microorganisms, and cultured cells. It catalyzes the final step of glycolysis, serving as one of the key rate-limiting enzymes in this process and a crucial enzyme for ATP production. Therefore, determining PK activity is of significant importance.Assay PrinciplePK catalyzes the conversion of Phosphoenolpyruvate (PEP) and ADP to ATP and Pyruvate. Lactate Dehydrogenase (LDH) further catalyzes the reaction between NADH and Pyruvate to produce Lactate and NAD⁺. The rate of decrease in NADH absorbance at 340 nm is measured, which reflects PK activity.Component50TStorageExtraction Buffer60 mL2-8℃Reagent A50 mL2-8℃Reagent B 2EA-20℃Reagent C25 µL×22-8℃Required Materials and Equipment (Not Provided)UV spectrophotometer, benchtop centrifuge, water bath, adjustable pipettes, 1 ml quartz cuvette, mortar and pestle, ice, and distilled water.Sample Preparation1.Bacteria or Cultured Cells:Collect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant.Add Extraction Buffer at a ratio of 1 ml per 5-10 million cells (e.g., 1 ml for 5 million cells/bacteria).Sonicate on ice (20% power or 200W, pulse 3s on/10s off, repeat 30 times).Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.2.Tissues:Homogenize tissue on ice in Extraction Buffer at a ratio of 1:5-10 (w/v) (e.g., 0.1 g tissue in 1 ml buffer).Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for assay.3.Serum (or Plasma) Samples:Assay directly.Assay Procedure1.Preheat the spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. Zero the instrument with distilled water.2.Sample Assay:(1) Preparation of Working Reagent II (WR II): Just before use, dissolve the contents of one vial of Reagent B in 22.5 ml of Reagent A and 2.65 ml distilled water. Mix thoroughly. Incubate WR II at 37°C (for mammalian samples) or 25°C (for other species) in a water bath for 5 minutes. Prepare fresh for each use.(2) Preparation of Working Reagent III (WR III): Just before use, dissolve the contents of one tube of Reagent C in 1.5 ml distilled water. Mix thoroughly. Prepare fresh for each use.(3) Reaction Setup: In a 1 ml quartz cuvette, add:50 µl sample50 µl WR III900 µl WR IIMix thoroughly and immediately record the absorbance (A₁) at 340 nm at 20 seconds. Record the absorbance again (A₂) after 2 minutes and 20 seconds (140 seconds total). Calculate ΔA = A₁ - A₂.PK Activity CalculationGeneral Formula:PK Activity = [ΔA × Vₜₒₜₐₗ ÷ (ε × d) × 10⁹] ÷ (Vₛₐₘₚₗₑ ÷ Vₛₐₘₚₗₑₜₒₜₐₗ) ÷ TWhere:Vₜₒₜₐₗ = Total reaction volume = 0.000975 L (975 µl)ε = NADH molar extinction coefficient = 6220 L/mol/cmd = Light path length = 1 cm10⁹ = Conversion factor from moles to nanomoles (nmol)T = Reaction time = 2 minVₛₐₘₚₗₑ = Sample volume added to reaction = 0.050 mlVₛₐₘₚₗₑₜₒₜₐₗ = Total volume of extract used = 1 ml (for tissues/cells)Cpr = Sample protein concentration (mg/ml)W = Sample mass (g)500 = Cell/Bacteria count (in millions, for the example calculation: 5 million = 500 × 10⁴)1. For Serum (Plasma):Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per ml of serum.Calculation:PK Activity (nmol/min/ml) = [ΔA × 0.000975 ÷ (6220 × 1) × 10⁹] ÷ 0.050 ÷ 2Simplified Formula: PK (nmol/min/ml) = 2613 × ΔA2. For Tissues, Bacteria, or Cells:a. Based on Sample Protein Concentration:* Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per mg of protein.* Calculation:PK Activity (nmol/min/mg prot) = [ΔA × 0.000975 ÷ (6220 × 1) × 10⁹] ÷ (0.050 × Cpr) ÷ 2Simplified Formula: PK (nmol/min/mg prot) = 2613 × ΔA ÷ Cprb. Based on Sample Fresh Weight:* Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per gram of tissue.* Calculation:PK Activity (nmol/min/g fresh weight) = [ΔA × 0.000975 ÷ (6220 × 1) × 10⁹] ÷ (W × 0.050 / 1) ÷ 2Simplified Formula: PK (nmol/min/g fresh weight) = 2613 × ΔA ÷ Wc. Based on Bacterial or Cell Density:* Definition: One unit of activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per 10⁴ cells.* Calculation (using the example of 5 million cells extracted in 1 ml):PK Activity (nmol/min/10⁴ cell) = [ΔA × 0.000975 ÷ (6220 × 1) × 10⁹] ÷ (5 × 0.050 / 1) ÷ 2Simplified Formula: PK (nmol/min/10⁴ cell) = 5.226 × ΔA... Read More | Inquire | Calcium, the most abundant mineral in the human body, is a crucial intracellular element that is responsible for regulating many physiological and pathological processes. Calcium is found in either the free ion form or in bound complexes, for example the calcium phosphate and calcium carbonate Calcium, the most abundant mineral in the human body, is a crucial intracellular element that is responsible for regulating many physiological and pathological processes. Calcium is found in either the free ion form or in bound complexes, for example the calcium phosphate and calcium carbonate complexes that make up bone tissue. Numerous physiological processes, including muscle contraction, cell adhesion, hormones/ neurotransmitters release, glycogen metabolism, cell proliferation/differentiation, blood clotting, nerve or synapthetic impulse transmission, and structural support of the skeleton are regulated by calcium signaling. Defects in the integrity of cell-specific calcium signaling systems may be associated with certain human diseases.Calcium Colorimetric Assay kit has been used to measure calcium concentration in hippocampal samples and MC3T3-E1 mouse osteoblast cell line, which were cultured in osteogenic induction medium... Read More | Product introduction：Dualucif The firefly & Renilla assay kit (dual luciferase reporter assay kit) provides an effective means to detect the expression of genes. In DLR detection, the activities of firefly luciferase and Renilla luciferase can be detected in a single sample in turn. FirstProduct introduction：Dualucif The firefly & Renilla assay kit (dual luciferase reporter assay kit) provides an effective means to detect the expression of genes. In DLR detection, the activities of firefly luciferase and Renilla luciferase can be detected in a single sample in turn. First, luciferin was used as substrate to detect the activity of firefly luciferase, then substances inhibiting the catalysis of firefly luciferase were added, and coelenterazine was added to detect the activity of Renilla luciferase to achieve dual luciferase reporter gene detection. The bioluminescence system of luciferase and its substrate can detect gene expression very sensitively and efficiently. Usually, the transcriptional regulatory element or 5'promoter region of the gene of interest is cloned upstream of luciferase, or the 3'-utr region is cloned downstream of luciferase to construct a reporter gene plasmid, and then transfect the cells. After the cells are treated with appropriate drugs, the cells are lysed, and the transcriptional regulation effect of drug treatment on the target gene is judged by detecting the luciferase activity. Renilla luciferase is more often used as an internal reference for detecting transfection efficiency to eliminate the difference in cell number and transfection efficiency. Firefly luciferase is a protein with a molecular weight of about 61 kDa. In the presence of ATP, magnesium ions and oxygen, it can catalyze the production of oxyluciferin from luciferin. In the process of luciferin oxidation, it will produce a light signal. Renilla luciferase is a protein with a molecular weight of about 36 kDa. In the presence of oxygen, it can catalyze the oxidation of coelenteramide to coelenteramide, and also produce light signals in the process of coelenteramide oxidation. The optical signal of this kit can be measured by chemiluminescence instrument, microplate reader or liquid scintillation tester. The kit has the characteristics of rapid detection, high sensitivity, wide detection range and no interference of cell endogenous activity.Instruction：1.Cell lysis ( 1 ) Remove the medium and gently wash twice with PBS ( adherent cells can be operated directly, suspension cells need to be centrifuged to collect cells ). Add 1 × Lysis Buffer ( diluted component A with sterile water at 4 : 1 ) according to the following scheme, and then place the culture plate on a micro-oscillator at room temperature for 15 min to fully lyse the cells. Note : The pyrolysis products can be stored at room temperature for 6 h, and can be stored at − 70 °C for a long time ( the pyrolysis products cannot be repeatedly frozen and thawed ). ( 2 ) The pyrolysis products were centrifuged at 10000-15000 rpm for 3-5 min. After centrifugation, the supernatant was transferred into a new EP tube for subsequent detection. Note : Cells can be detected immediately after lysis, or frozen, and re-detected when needed. The frozen samples need to be thawed to room temperature for detection. 2. Preparation of working fluid ( 1 ) Restore all components to room temperature. ( 2 ) Dilute component C with component B to 0.2 mg / mL firefly luciferase working solution. Note : The firefly luciferase working solution cannot be repeatedly frozen and thawed. If the amount of a single experiment is small, it is recommended to be subpackaged into small specifications according to a single amount of use. ( 3 ) The E component was diluted into the renilla luciferase working solution with the D component, and the dilution method was 1 µL E component was added to the 49 µL D component. Note : Renilla luciferase working solution needs to be prepared now. 3.chemiluminescence value detection ( 1 ) According to the operation instructions of the instrument, the instrument with chemiluminescence detection function was opened, such as multifunctional microplate reader. The parameters were set, the determination time was 10 s, and the determination interval was 2 s. ( 2 ) each sample determination, take the sample 20-100 µL ( if the sample volume is enough, please add 100 µL ; if the sample amount is insufficient, the amount can be appropriately reduced, but the amount of detection holes needs to be consistent ). 1 × Lysis Buffer was blank control. ( 3 ) 100 µL firefly luciferase working solution was added to determine the RLU ( relative light unit ) value ( it is recommended that the microplate reader set up the Shaking mixing function ). Note : Since the luminescence is instantaneous, it is recommended to detect immediately after adding the firefly luciferase working solution. ( 4 ) 100 µL renilla luciferase working solution was added to determine the RLU ( relative light unit ) value ( Shaking mixing function is recommended for microplate reader ). ( 5 ) In the case of renilla luciferase as an internal reference, the RLU value measured by firefly luciferase was divided by the RLU value measured by renilla luciferase. According to the obtained ratio, the activation degree of the target reporter gene between different samples was compared. If firefly luciferase is used as an internal reference, similar calculations can also be performed.Component：Recommendation：It is recommended to use component B in advance to prepare 2 mg / mL storage solution, component B, component D and component C prepared as storage solution, and to carry out small batch packing according to the experimental requirements. All test working fluids are recommended to be used now to avoid repeated freezing and thawing.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. in order to obtain the best determination effect, when using a single tube chemiluminescence instrument for determination, the time from the mixing of sample and determination reagent to the pre determination should be controlled as much as possible; When using a multi-functional fluorescent microplate reader with chemiluminescence detection function, it is advisable to add all samples first, and then uniformly add firefly luciferase detection reagent. 3. the strongest wavelength of firefly luciferase catalyzed bioluminescence is 560 nm, and the strongest wavelength of Renilla luciferase catalyzed bioluminescence is 480 nm. 4. to prevent interference between holes, it is recommended to use white opaque orifice plate. 5. due to the influence of temperature on enzyme reaction, the sample and reagent should be measured after reaching room temperature. 6. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Study on gene expression regulation and promoter... Read More | Product contentS666146Component50 T200 TStorageS666146ABuffer GR25 mL120 mLRTS666146BBuffer GL25 mL120 mLRTS666146CBuffer GW1 (concentrate)13 mL52 mLRTS666146DBuffer GW2 (concentrate)15 mL75 mLRTS666146EBuffer GE15 mL60 mLRTS666146FProteinase K1.25 mL4×1.25 mLRTS666146GSpin Columns DS with Product contentS666146Component50 T200 TStorageS666146ABuffer GR25 mL120 mLRTS666146BBuffer GL25 mL120 mLRTS666146CBuffer GW1 (concentrate)13 mL52 mLRTS666146DBuffer GW2 (concentrate)15 mL75 mLRTS666146EBuffer GE15 mL60 mLRTS666146FProteinase K1.25 mL4×1.25 mLRTS666146GSpin Columns DS with Collection Tubes50 sets 200 setsRTS666146HCentrifuge Tubes (1.5 mL)50 EA200 EARTProductsThis kit provides a simple and rapid method for the isolation and purification of total DNA from buccal swab samples. The kit adopts a silica matrix membrane that can specifically bind DNA and a unique buffer system to adsorb DNA efficiently and specifically, and 0.5-3.5 µg of genomic DNA can be obtained from each swab, and the extracted DNA fragments are large, pure and of stable and reliable quality. It is suitable for enzyme digestion, PCR, library construction, Southern hybridization and other experiments.Self-contained reagent: anhydrous ethanol.Pre-experiment Preparation and Important Notes1. 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.2. If precipitation is found in Buffer GL before use, dissolve Buffer GL in a 56°C water bath.3. All centrifugation steps can be performed at room temperature.4. Sampling: Use a buccal swab to wipe the inside of the mouth 6 times, dry for 2 hours and store. To ensure that the sample is not contaminated by food or drink, do not eat or drink for 30 minutes before sampling.Procedure1. The swab of the buccal swab was cut from the rod with scissors and placed in a 2mL centrifuge tube (supplied) and 400µL Buffer GR was added.Note: For genomic DNA without RNA contamination, add 4 µL of RNase A solution at a concentration of 100 mg/ml and shake to mix.2. Add 20 µL of Proteinase K and 400 µL of Buffer GL, immediately vortex and shake for 15 seconds and mix thoroughly.Note: Mix well immediately after adding Buffer GL; do not add Proteinase K directly to Buffer GL for use.3.56°C for 10 minutes and centrifuge briefly so that the solution on the walls of the tube collects at the bottom.4. Add 400 µL of anhydrous ethanol, vortex and shake to mix thoroughly, and centrifuge briefly so that the solution on the wall of the tube collects at the bottom of the tube.Note: The addition of anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.5. Add the solution and precipitate obtained in the previous step to the Spin Columns DS in two batches of up to 700 µL at a time into the collection tube. centrifuge the column at 12,000 rpm (∼13,400 × g) for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.6. Add 500 µL of Buffer GW1 to the adsorbent 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 adsorbent 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 the column at 12,000 rpm for 3 minutes, pour off the waste liquid in the collection tube, and put the 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 1 minute 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 1.5 mL centrifuge tube, add 50 µL of Buffer GE or sterilized water to the middle of the adsorption column overhanging the column, let stand at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store at -20℃.Attention:(1) If the downstream experiment is sensitive to pH or EDTA, it can be eluted with sterilized water. The pH value of the eluent has a great influence on the elution efficiency. If the eluent is made of water, the pH value should be 7.0-8.5 (the pH value of water can be adjusted to this range by using NaOH), and the elution efficiency is not high when the pH value is lower than 7.0.2) For long-term storage, it is recommended to elute with Buffer GE and store at -20°C... Read More |