| Description | Glucokinase (GK, EC 2.7.1.2) is a member of the hexokinase family, primarily found in mature hepatocytes and pancreatic islet cells. Under normal physiological conditions, the main role of GK is to monitor blood glucose levels. Detection Principle: Glucokinase (GK) phosphorylates glucose to produce Glucokinase (GK, EC 2.7.1.2) is a member of the hexokinase family, primarily found in mature hepatocytes and pancreatic islet cells. Under normal physiological conditions, the main role of GK is to monitor blood glucose levels. Detection Principle: Glucokinase (GK) phosphorylates glucose to produce glucose-6-phosphate. This product is further coupled with glucose-6-phosphate dehydrogenase and NADP⁺. The increase in NADPH absorbance at 340 nm is measured, allowing for the calculation of the enzyme's activity.Component100TStorageExtraction Buffer120 mL2-8℃Reagent 120 mL2-8℃Reagent 21EA-20℃Reagent 31EA2-8℃Reagent 2 (Powder, 1 vial) Preparation:Before use, centrifuge at 8000 g, 4°C for 2 minutes to collect the powder at the bottom of the tube (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 3 (Powder, 1 vial) Preparation:Before opening, ensure the powder is at the bottom (can be flicked manually).Add 18 mL of Reagent 1 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, Distilled water (Deionized water or Ultrapure water are also 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 Extraction1.1 Tissue SamplesWeigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize in an ice bath. Centrifuge at 12,000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.Note: If increasing the sample amount, maintain a tissue mass (g) to Extraction Buffer volume (mL) ratio between 1:5 and 1:10.1.2 Bacterial/Cell SamplesCollect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer per 5 million bacteria/cells. Disrupt the bacteria or cells by sonication in an ice bath (power 20% or 200W, pulse 3s on, 10s off, repeat 30 times). Centrifuge at 12,000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.Note: If increasing the sample amount, maintain a bacteria/cell count (10⁴) to Extraction Buffer volume (mL) ratio between 500:1 and 1000:1.1.3 Liquid SamplesAssay directly. If turbid, centrifuge and use the supernatant for assay.2. Assay Steps2.1 Preheat the microplate reader for at least 30 minutes. Set the wavelength to 340 nm.2.2 Pre-warm the prepared Reagent 2 and Reagent 3 at 25°C for 5 minutes to reach room temperature.2.3 Add reagents sequentially to a 96-well plate:ReagentTest Well (µL)Sample20Reagent 210Reagent 3170Mix thoroughly. Read the absorbance at 340 nm at 1 minute (A₁) and again at 21 minutes (A₂, i.e., after 20 minutes of reaction). Calculate ΔA = A₂ - A₁.Note:If ΔA is close to zero, the reaction time can be appropriately extended to 30 minutes or longer 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; the new sample volume (V₁) must then be substituted into the calculation formula.If the increase trend is unstable, read the absorbance every 10 seconds and select a linearly increasing time period for calculation. The corresponding A values for this period should be used to calculate ΔA and substituted into the formula.3. Calculation of Results3.1 Based on Sample Protein ConcentrationUnit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of NADPH per minute per mg of tissue protein.Derived Formula: GK (nmol/min/mg prot) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (V₁ × Cpr) ÷ TSimplified Formula: GK (nmol/min/mg prot) = 160.77 × ΔA ÷ Cpr3.2 Based on Sample Fresh WeightUnit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of NADPH per minute per gram of tissue.Derived Formula: GK (nmol/min/g fresh weight) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (W × V₁ ÷ V) ÷ TSimplified Formula: GK (nmol/min/g fresh weight) = 160.77 × ΔA ÷ W3.3 Based on Bacterial or Cell DensityUnit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of NADPH per minute per 10⁴ bacteria or cells.Derived Formula: GK (nmol/min/10⁴ cells) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ (500 × V₁ ÷ V) ÷ TSimplified Formula: GK (nmol/min/10⁴ cells) = 0.32 × ΔA3.4 Based on Liquid VolumeUnit Definition: One unit of enzyme activity is defined as the amount that produces 1 nmol of NADPH per minute per mL of liquid.Derived Formula: GK (nmol/min/mL) = [ΔA ÷ (ε × d) × V₂ × 10⁹] ÷ V₁ ÷ TSimplified Formula: GK (nmol/min/mL) = 160.77 × ΔAParameter Definitions:ε: Molar extinction coefficient of NADPH (6.22 × 10³ L/mol/cm)d: Light path length for the 96-well plate (0.5 cm)V: Volume of Extraction Buffer added (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 (20 minutes)W: Sample weight (g)500: Total number of bacteria or cells (5 million)Cpr: Sample protein concentration (mg/mL); Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.PrecautionsIt 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 | Description：Acetylcholinesterases (AChEs) are enzymes that hydrolyze the neurotransmitter acetylcholine (ACh) to acetate and choline. AChE is located at the synaptic cleft and functions to terminate synaptic transmission by catalyzing the breakdown of ACh allowing cholinergic neurons to Description：Acetylcholinesterases (AChEs) are enzymes that hydrolyze the neurotransmitter acetylcholine (ACh) to acetate and choline. AChE is located at the synaptic cleft and functions to terminate synaptic transmission by catalyzing the breakdown of ACh allowing cholinergic neurons to return to a resting state after activation. It is also found in membranes of red blood cells, motor and sensory fibers, muscles, nerves and central and peripheral tissues. Changes in AChE activity may result from exposure to certain insecticides, which act as cholinesterase inhibitors. Inhibitors of AChE are also used to treat certain conditions such as dementia.Acetylcholinesterase activity assay kit has been used to determine the activity of acetylcholinesterase in a rat organophosphate model and in brain tissue homogenates.Principle：Acetylcholinesterase can catalyze the hydrolysis of acetylcholine to choline, and the reaction of choline with disulfide p-nitrobenzoic acid to produce 5-merhydryl-nitrobenzoic acid (TNB). The product has a characteristic absorption peak at 412 nm, and the activity of acetylcholinesterase can be characterized by the change of light absorption valueThe Dilution Calculator EquationConcentration (start)xVolume (start)= Concentration (final)× Volume (final)This equation is commonly abbreviated as: C1V1 = C2V2... Read More | Bacterial protein extraction reagents use mild non-ionic detergents and are suitable for extracting recombinant proteins expressed in Escherichia coli and insect cells. During the extraction process, there is no need for ultrasonic fragmentation, effectively avoiding contamination of exogenous Bacterial protein extraction reagents use mild non-ionic detergents and are suitable for extracting recombinant proteins expressed in Escherichia coli and insect cells. During the extraction process, there is no need for ultrasonic fragmentation, effectively avoiding contamination of exogenous proteins. This product can be applied to extract soluble proteins from bacterial lysates. The bacterial protein extraction kit adds a mixture of lysozyme, DNase I, and protease inhibitors to the extraction reagent, which can improve the efficiency of protein extraction and reduce the viscosity caused by DNA, effectively avoiding protein degradation. The extracted protein maintains biological activity and can be subjected to downstream operations such as IP, Western blot, and protein purification. Component B665764 100 preps Bacterial Protein Extraction Reagent 100 ml Protease Inhibitor Cocktail (100x) 1 ml Lysozyme （50 mg/ml） 200µl DNaseⅠ（1,000 U/ml） 100µl Notes:1. This product is suitable for extracting proteins from fresh or frozen bacterial and insect cells.2. This product uses Tris buffer system. Please use the same buffer system for protein purification after extraction.3. The protein lysis solution obtained from this product can be used for protein quantification using BCA or Bradford method.4. For special strains, if the extraction effect is not ideal, the sample can be frozen before protein extraction.5. Depending on the specific situation, protease inhibitors, salts, chelating agents, reducing agents, etc. can be added to this product.Operation steps: ● Insect cell protein extraction1. Collect cells by low-speed centrifugation. Add 10 to every 1 ml of Bacterial Protein Extraction Agent µ The Protein Inhibitor Cocktail is 1 x working fluid.2. Weigh the wet weight of the cells and add 1 x working solution at a rate of 10 ml/g.3. After resuspension, incubate on ice for 20 minutes (the ice storage time should be adjusted according to different cell types).Centrifuge at 4.15000 × g for 15 minutes to isolate soluble proteins. ● Extraction of soluble bacterial proteins 1. Centrifuge for 10 minutes at a rate of 5000 × g and collect the bacterial cells.2. Optional steps: Add 1 ml of Bacterial Protein Extraction Reagent every 1 ml µ DNase I (1000 U/ml), 2 µ Lysozyme (50 mg/ml) and 10 µ Protein Inhibitor Cocktail, vortex oscillation and mixing. 3. Add 20 ml of Bacterial Protein Extraction Reagent to each gram of bacterial precipitate, and add the extraction solution to the bacterial precipitate. Vortex thoroughly or use a pipette to blow up and down until the bacterial precipitate is completely resuspended.4. After resuspension, incubate at room temperature for 10-15 minutes (the storage time should be adjusted according to different cell types). 5. Centrifuge at 15000 × g for 5 minutes.6. Transfer the supernatant to a new centrifuge tube (the supernatant is soluble protein) for protein quantification and downstream experiments.Note: If the target protein exists in the form of inclusion bodies, inclusion body protein solution can be used for dissolution or expression conditions can be optimized to increase the expression of soluble proteins.Frequently asked questions: Problem Possible reasons Resolvent The target protein is insoluble The target protein is expressed as an inclusion body Optimize expression conditions or add Lysozyme and DNase I to protein extraction reagents using inclusion body protein solution After adding Lysozyme, the target protein has not been extracted yet Temperature too low Restore the reagent to room temperature After adding Lysozyme, the target protein has not been extracted yet Lysozyme Decreased or inactivated activity Add more Lysozymes or replace with new enzymes Extract has high viscosity DNase I Decreased or inactivated activity Add more DNase I or replace with a new DNase I to increase the final concentration of magnesium ions to 2 mM After protein extraction, most of the proteins still exist in the precipitate Excessive protein content Add Lysozyme and DNase I The protein extraction reagent has sediment precipitation Temperature too low Restore the protein extraction reagent to room temperature... Read More | Inquire | Product contentY666144Component50 TStorageY666144ABuffer P115 mLRTY666144BBuffer P215 mLRTY666144CBuffer N320 mLRTY666144DBuffer PS15 mLRTY666144EBuffer PB10 mLRTY666144FBuffer PW (concentrate)10 mLRTY666144GBuffer EB10 mLRTY666144HGlass Beads2 gRTY666144IRNase A (10mg/mL)150 µLRTY666144JSpin Product contentY666144Component50 TStorageY666144ABuffer P115 mLRTY666144BBuffer P215 mLRTY666144CBuffer N320 mLRTY666144DBuffer PS15 mLRTY666144EBuffer PB10 mLRTY666144FBuffer PW (concentrate)10 mLRTY666144GBuffer EB10 mLRTY666144HGlass Beads2 gRTY666144IRNase A (10mg/mL)150 µLRTY666144JSpin Columns DM with Collection Tubes50 setsRTProductsThis kit is improved on the basis of common alkaline lysis method, the glass beads can effectively break the yeast cell wall, the new silica matrix membrane and buffer system can efficiently and specifically bind the plasmid DNA, and at the same time can maximize the removal of proteins and other impurities, the whole process is convenient and fast, no need to use toxic and harmful reagents, and can be processed at the same time for multiple samples. In addition to yeast cells, it can also be used in E. coli. Plasmid DNA extracted with this kit can be used in various molecular biology experiments, such as ligation, transformation, sequencing and library screening.Self-contained reagents: β-mercaptoethanol, anhydrous ethanol.Pre-experiment Preparation and Important Notes1. All components can be stably stored in dry, room temperature (15-30℃) environment for 1 year, the adsorption column can be stored at 2-8℃ for a longer period of time, and Buffer P1 with RNase A can be stably stored at 2-8℃ for 6 months.2. Before the first use, add all the RNase A solution to Buffer P1, mix well, and store at 2-8℃.3. Anhydrous ethanol should be added to Buffer PW before first use according to the instructions on the reagent bottle label.4. Before use, please check whether Buffer P2 and Buffer N3 are crystallized or precipitated. If there is any crystallization or precipitation phenomenon, it can be clarified by taking a water bath at 37℃ for a few minutes to restore the clarity.5. Be careful not to touch Buffer P2 and Buffer N3 directly, and tighten the lid immediately after use.6. The amount of plasmid extracted is related to the yeast strain, plasmid copy number, culture conditions, etc. Usually, yeast plasmid copy number is very low, which is difficult to be detected by electrophoresis or spectrophotometer method.Procedure1. Take 1-5 ml of yeast culture (maximum 5×107 yeast cells, generally for Saccharomyces cerevisiae OD = 1.0, equivalent to 1-2×107 cells/ml) and add it to a centrifuge tube (self-provided), centrifuge for 30 seconds at 12,000 rpm (~13,400×g), collect the bacterial precipitate, and aspirate as much as possible to discard the supernatant.2. Add 250µl Buffer P1 to the bacterium (please check if RNase A has been added first) and resuspend the precipitate.3. Add 40mg of Glass Beads to the above mixture and vortex and shake for 10 minutes.4. Add 250 µl of Buffer P2 to the centrifuge tube, mix gently by turning up and down 6-8 times, and let stand at room temperature for 5-10 minutes, at which time the bacterial solution should become clear and viscous.Note: Mix gently, do not shake violently, so as not to interrupt the genomic DNA, resulting in genomic DNA fragments mixed in the extracted plasmid. If the solution does not become clear, it suggests that the amount of bacteria may be too large and the lysis is not complete, and the amount of bacteria should be reduced.5. Add 350 µl of Buffer N3 to the centrifuge tube and immediately mix gently up and down 6-8 times, at which point a white flocculent precipitate appears, and centrifuge at 12,000 rpm for 20 minutes.Note: Buffer N3 should be mixed immediately after addition to avoid localized precipitation.6. Column Equilibration: Add 200 µl of Buffer PS to the Spin Columns DM in the collection tube, centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and place the column back into the collection tube.7. Add the supernatant from step 5 to the adsorbent column that has been loaded into the collection tube, taking care not to aspirate the precipitate.Note: The maximum volume of the adsorption column is 750 µl, and the solution is passed through the column in 2 times.8. Centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube and place the adsorption column back into the collection tube.9. Add 150 µl Buffer PB to the adsorbent column, centrifuge at 12,000 rpm for 1 min, pour off the waste liquid in the collection tube, and put the adsorbent column back into the collection tube.10. Add 750 µl Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first), centrifuge at 12,000 rpm for 1 minute, and pour off the waste liquid in the collection tube.11. Place the column back into the recovery collection tube and centrifuge at 12,000 rpm for 2 minutes, pouring off the waste liquid. Leave the 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; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).12. Place the adsorbent column in a new centrifuge tube, add 50-100 µl of Buffer EB to the center of the adsorbent membrane dropwise, let it stand at room temperature for a few minutes, centrifuge at 13,000 rpm for 1 minute, and collect the plasmid solution into the centrifuge tube. Store the plasmid at -20°C.Attention:1) To increase the recovery efficiency of the plasmid, the resulting solution can be reintroduced into the adsorbent column, left at room temperature for a few minutes, centrifuged at 13,000 rpm for 1 minute, and the plasmid solution collected into a centrifuge tube.2) When the plasmid copy number is low or >10 kb, Buffer EB is preheated at 65-70°C in a water bath, which can increase the extraction efficiency.3) Usually yeast plasmids have very low copy number and are difficult to detect by electrophoresis or spectrophotometry. If the extracted plasmid is to be used in the next step of the experiment, it is usually recommended to use 1-5µl of the plasmid as PCR template, and 5-10µl of the plasmid for transformation of E. coli.4) Commercial high transformation efficiency receptor cells should be used for transformation of E. coli... Read More |