| Description | Animal liver and kidneys are the main organs for amino acid metabolism. Therefore, changes in urinary amino acids best reflect the physiological state of the liver and kidneys. Additionally, amino acid levels can indicate conditions such as burns and typhoid fever. The amino acid content in plants Animal liver and kidneys are the main organs for amino acid metabolism. Therefore, changes in urinary amino acids best reflect the physiological state of the liver and kidneys. Additionally, amino acid levels can indicate conditions such as burns and typhoid fever. The amino acid content in plants is significant for studying nitrogen metabolism changes under different conditions and during various growth and development stages, as well as for understanding nitrogen absorption, transport, assimilation, and nutritional status in plants.Detection Principle: The α-amino group of amino acids reacts with ninhydrin hydrate to produce a blue-purple compound with a characteristic absorption peak at 570 nm. The amino acid content is calculated by measuring the absorbance at 570 nm.Detection Range: 0.625 - 40 µmol/mLSensitivity: 0.5 µmol/mLApplicable Samples: Serum (plasma), animal/plant tissues, cells, cell culture supernatants, bacteria, urineG1501758Component96TStorageG1501758AExtraction Buffer100 mL2-8℃G1501758BAssay Buffer10 mL2-8℃G1501758CSubstrate1EA2-8℃. Store in the dark.G1501758DStandard (10mg Cysteine)1EA2-8℃. Store in the dark.User-Prepared Instruments and ReagentsMicroplate reader or visible spectrophotometer (capable of measuring absorbance at 570 nm)96-well plate or micro glass cuvettes, adjustable micropipettes and tipsRefrigerated centrifuge, water bathDeionized water, EthanolHomogenizer (for tissue samples)Experimental Procedure1. Reagent PreparationReagent NameReagent PreparationNotesExtraction BufferReady-to-use; Equilibrate to room temperature before use.Store at 4°C.Assay BufferReady-to-use; Equilibrate to room temperature before use.Store at 4°C. Toxic and irritant. Perform experiments in a fume hood.SubstrateToxic and irritant. Perform experiments in a fume hood.Working SubstratePrepare before use: Dissolve in 4 mL of 95% Ethanol.Unused dissolved substrate can be stored at 4°C protected from light for one week. For long-term storage, aliquot and store at -20°C protected from light for one month. Avoid repeated freeze-thaw cycles.StandardPrepare before use: Add 2.066 mL deionized water to dissolve completely, resulting in a 40 µmol/mL stock.Unused dissolved standard can be stored at 4°C for one week. For long-term storage, aliquot and store at -20°C for one month. Avoid repeated freeze-thaw cycles.2. Standard Curve SetupDilute the 40 µmol/mL standard stock solution with deionized water to concentrations of 20, 10, 5, 2.5, 1.25, and 0.625 µmol/mL as shown in the table below.TubeStandard VolumeDeionized Water Volume (µL)Standard Concentration (µmol/mL)Std.1200µL of 40µmol/mL040Std.2100µL of Std.110020Std.3100µL of Std.210010Std.4100µL of Std.31005Std.5100µL of Std.41002.5Std.6100µL of Std.51001.25Std.7100µL of Std.61000.625Note: The standard curve must be generated with each experiment. Diluted standard solutions are unstable and must be used within 4 hours.3. Sample PreparationNote: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to one month.3.1 Animal TissueWeigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and homogenize thoroughly at room temperature. Transfer the homogenate to a 1.5 mL microcentrifuge tube. Cap tightly (to prevent moisture loss) and incubate in a boiling water bath for 15 minutes. Cool with tap water. Centrifuge at 10,000 rpm for 10 minutes at room temperature. Collect the supernatant for assay.3.2 Plant TissueWeigh approximately 0.1 g of tissue. Add 1 mL of Extraction Buffer and grind. Sonicate for 5 minutes at room temperature (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Transfer to a 1.5 mL microcentrifuge tube. Cap tightly (to prevent moisture loss) and incubate in a boiling water bath for 15 minutes. Cool with tap water. Centrifuge at 10,000 rpm for 10 minutes at room temperature. Collect the supernatant for assay.3.3 Cells or BacteriaCollect 5 million cells or bacteria into a centrifuge tube. Wash cells with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Sonicate for 5 minutes at room temperature (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Transfer to a 1.5 mL microcentrifuge tube. Cap tightly (to prevent moisture loss) and incubate in a boiling water bath for 15 minutes. Cool with tap water. Centrifuge at 10,000 rpm for 10 minutes at room temperature. Collect the supernatant for assay.3.4 Serum (Plasma), Cell Culture Supernatant, Urine, and Other LiquidsPipette 0.5 mL of the liquid sample and add 0.5 mL of Extraction Buffer. Cap tightly (to prevent moisture loss) and incubate in a boiling water bath for 15 minutes. Cool with tap water. Centrifuge at 10,000 rpm for 10 minutes at room temperature. Collect the supernatant for assay.Note: If protein concentration measurement is required, use Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648).4. Assay Steps4.1 Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 570 nm. For spectrophotometers, zero the instrument with deionized water.4.2 Sample Measurement (Add reagents sequentially into microcentrifuge tubes as below):ReagentBlank Tube (µL)Standard Tube (µL)Test Tube (µL)Deionized Water1000Standard (various conc.)0100Sample0010Working Substrate202020Assay Buffer5050504.3 Mix well and cap the tubes tightly (to prevent moisture loss). Incubate in a boiling water bath for 5 minutes. Cool in tap water for 10 seconds. Add 120 µL of 60% ethanol to each tube and invert several times to mix. Transfer 150 µL from each tube to the corresponding wells of a 96-well plate or micro glass cuvette. Measure the absorbance at 570 nm, recorded as A blank, A standard, and A test. Calculate ΔA test = A test - A blank and ΔA standard = A standard - A blank (The blank tube only needs to be prepared once). All measurements must be completed within 30 minutes after color development. Note:It is recommended to perform a preliminary test with 2-3 samples expected to have significant differences before the formal experiment. If A <sub> test </sub> is greater than 2.0, dilute the sample further with deionized water and multiply the result by the dilution factor.Proline and hydroxyproline do not produce an absorption peak at 570 nm when reacting with ninhydrin. Therefore, the results measured at 570 nm do not include these two amino acids.5. Calculation of ResultsNote: We provide two formulas, including the derived formula and a simplified version. They are equivalent. It is recommended to use the simplified formula in bold for final calculation.5.1 Standard Curve PlottingPlot the standard concentration (y-axis) against ΔA standard (x-axis) to generate the standard curve. Substitute ΔA test into the standard curve equation to calculate y (µmol/mL).5.2 Sample Amino Acid Content Calculation(1) Based on Sample WeightAmino Acid Content (µmol/g weight) = y ÷ (W ÷ V<sub>extraction</sub>) × n = y ÷ W × n(2) Based on Protein ConcentrationAmino Acid Content (µmol/mg prot) = y ÷ Cpr × n(3) Based on Bacterial or Cell CountAmino Acid Content (µmol/10⁴ cells) = y ÷ (Count ÷ V<sub>extraction</sub>) × n = y ÷ 500 × n = 0.002 × y × n(4) Based on Liquid VolumeAmino Acid Content (µmol/mL) = y × 2 × nParameter Definitions:W: Sample weight (g)V extraction : Volume of Extraction Buffer added (1 mL)n: Sample dilution factorCpr: Protein concentration of the supernatant (mg/mL)500: Total number of bacteria or cells (5 million)2: Dilution factor for liquid samples [(0.5 mL sample + 0.5 mL Buffer) / 0.5 mL sample = 2]6. Representative ResultsTypical Standard Curve: y = 20.349x - 0.423, R² = 0.9971 Figure 1: Total Amino Acid Standard Curve Precautions1. Biochemical reagents are generally irritating, biologically toxic, etc. For your safety and health, please use appropriate biosafety precautions throughout the experiment, including wearing lab coats, masks, gloves, head covers, etc. Perform experiments in a fume hood or biosafety cabinet.2. This product is for research use only. Not for use in clinical diagnosis... 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 | DescriptionThe Baran Late-Stage Toolkit is a convenient collection of 12 highly innovative reagents that are highly effective in the diversification of complex molecules. The contents in the box are 11 Baran Diversinates™and one vial of Palau′Chlor®in amounts of 100 mg each. For DescriptionThe Baran Late-Stage Toolkit is a convenient collection of 12 highly innovative reagents that are highly effective in the diversification of complex molecules. The contents in the box are 11 Baran Diversinates™and one vial of Palau′Chlor®in amounts of 100 mg each. For obtaining larger amounts of any desired kit component, see the kit component table at the bottom of the page.Useful Topics:Late Stage FunctionalizationBaran Group – Professor Product PortalPalau′ChlorDiversinates... Read More | Product contentN666081Component50 TStorageN666081ANc-Buffer A50 mL2-8℃N666081BNc-Buffer B3 mL2-8℃N666081CNc-Buffer C25 mL2-8℃N666081DProtease Inhibitor Cocktail750 µL-20℃. Avoid freeze/thaw cycle.ProductsThe Nc-Nucleus/Plasma Protein Extraction Kit is a simple and rapid Product contentN666081Component50 TStorageN666081ANc-Buffer A50 mL2-8℃N666081BNc-Buffer B3 mL2-8℃N666081CNc-Buffer C25 mL2-8℃N666081DProtease Inhibitor Cocktail750 µL-20℃. Avoid freeze/thaw cycle.ProductsThe Nc-Nucleus/Plasma Protein Extraction Kit is a simple and rapid method for extracting nucleus and plasma proteins from mammalian cells and tissues, and the extracted proteins remain biologically active. The kit first cleaves the cell membrane and releases plasma proteins using the plasma protein extraction reagent, and then centrifuges the nucleus to obtain a nucleus precipitate. Finally, the nuclear proteins are extracted by the nuclear protein extraction reagent. The extracted nuclear and plasma proteins are of high purity, effectively avoiding cross-contamination of nuclear and plasma proteins, and can be used for subsequent operations such as Western, Gel Shift, reporter gene detection and enzyme activity determination.Caveat1. If phosphorylated proteins are to be extracted, add a phosphatase inhibitor to the extraction reagent.2. All sample handling should be done on ice.3. The amount of reagents can be adjusted according to the specific experimental situation to ensure that the ratio of each reagent used is Nc-Buffer A:Nc-Buffer B:Nc-Buffer C = 100:5.5:50.4. Higher speeds can be used for centrifugation.ProcedureI Extraction of cytoplasmic and cytosolic proteins from cells1. Please remove the extraction reagents Nc-Buffer A and Nc-Buffer C for pre-cooling before protein extraction.2. Collect the cells and count them. Centrifuge to remove supernatant.3. 1×107 cells were added with 1 ml of Nc-Buffer A (added to Protease Inhibitor Cocktail at a ratio of 1:99 within 2-3 minutes prior to protein pumping), vortexed for 5 seconds to mix well, and incubated on ice for 20 minutes.Note: The characteristics of various cells are different, and the amount of Nc-Buffer A needs to be adjusted according to the characteristics of different cells. If the protein concentration is small, reduce the amount of Nc-Buffer A and subsequent Nc-Buffer B and Nc-Buffer C proportionally.4. Add 55 µl of Nc-Buffer B, vortex for 5 seconds to mix thoroughly, and incubate on ice for 1 minute.5. Centrifuge at 12,000 rpm (~13,400 x g) for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is cytoplasmic protein).6. Add 500 µl of Nc-Buffer C (add Protease Inhibitor Cocktail at a ratio of 1:99 before use) to the precipitate obtained in the previous step, vortex for 5 seconds to mix thoroughly, resuspend the precipitate and incubate on ice for 40 minutes, vortexing and mixing at 10-minute intervals for about 15-30 seconds each time.7. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is for cytosolic proteins).II Extraction of cytoplasmic and cytosolic proteins from tissues1. Sampling and preservation of tissues.2. Remove the extraction reagents Nc-Buffer A and Nc-Buffer C for pre-cooling before protein extraction.3. Weigh the tissue and add 1 ml of Nc-Buffer A per 100 mg of tissue (add Protease Inhibitor Cocktail 2-3 minutes before protein extraction at a ratio of 1:99), homogenize well on ice with a homogenizer, and incubate on ice for 20 minutes.Note: The characteristics of various tissues are different, and the amount of Nc-Buffer A needs to be adjusted according to different tissues. If the protein concentration is small, reduce the amount of Nc-Buffer A and subsequent Nc-Buffer B and Nc-Buffer C proportionally.4. Add 55 µl of Nc-Buffer B, vortex for 5 seconds to mix thoroughly, and place on ice for 1 minute of incubation.5. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is cytoplasmic protein).6. Add 500 µl of Nc-Buffer C (add Protease Inhibitor Cocktail at a ratio of 1:99 before use) to the precipitate obtained in the previous step, vortex for 5 seconds to mix thoroughly, resuspend the precipitate and incubate on ice for 40 minutes, vortexing and mixing at 10-minute intervals at, each time for about 15-30 seconds.7. Centrifuge at 12,000 rpm for 15 minutes at 4°C, collect the supernatant (as clean as possible) into a new centrifuge tube and store at -20°C (this extract is cytosolic protein)... Read More | Q665720 Component 200T Storage Q665720A Buffer L2 25 mL RT Q665720B Buffer N3 80 mL RT Q665720C Buffer PB 35 mL RT Q665720D Buffer PW (concentrate) 25 mL RT Q665720E Buffer EB 30 mL RT Q665720F RNase A (10 mg/mL) 800 渭L RT Q665720G Spin Columns DM with Collection Tubes 200 EA RTProduct Q665720 Component 200T Storage Q665720A Buffer L2 25 mL RT Q665720B Buffer N3 80 mL RT Q665720C Buffer PB 35 mL RT Q665720D Buffer PW (concentrate) 25 mL RT Q665720E Buffer EB 30 mL RT Q665720F RNase A (10 mg/mL) 800 渭L RT Q665720G Spin Columns DM with Collection Tubes 200 EA RTProduct IntroductionThe biggest feature of this kit: simple and fast, high extraction volume. The whole extraction process does not take more than 10 minutes, without centrifugation to collect bacteria and resuspend the bacterium, directly add the unique super lysate Buffer L2 to the cultured bacterial solution, followed by neutralization, centrifugation and passing through the column, and the extracted plasmid can be as high as 30 µg, and maximize the removal of proteins, genomes and other impurities. The extracted plasmid DNA can be directly used for bacterial transformation, digestion, PCR, in vitro transcription, sequencing and other downstream experiments.Self-contained reagent: anhydrous ethanol.Pre-experiment Preparation and Important Notes1. The kit can be stored in a dry, room temperature (15-30°C) environment for 1 year. For longer storage, the centrifuge columns can be placed at 2-8°C.2. Before the first use, add all of the RNase A solution to Buffer N3, mix well, and store at 2-8°C.3. Anhydrous ethanol should be added to Buffer PW before the first use according to the instructions on the reagent bottle label.4. If there is any precipitation in Buffer L2 before use, please put it in a 37℃ water bath and keep mixing until the solution becomes clear before use.Operation steps1. Take 600 µl of bacterial culture into a 1.5 ml centrifuge tube (supplied).2. Add 100 µl of Buffer L2 to the above centrifuge tube and gently turn the solution up and down 8 times; the solution should change from turbid to a clear purple color, indicating complete lysis. The cleavage time should not exceed 2 minutes.3. Add 350 µl of Buffer N3 to the above centrifuge tube (please check that RNaseA has been added first) and immediately mix well by turning up and down about 8-10 times, at which point the solution should turn completely yellow and a yellow precipitate should form. centrifuge at 13,000 rpm for 2-3 minutes.4. Slowly pour the supernatant obtained in step 3 into the prepared adsorption columns (Spin Columns DM with Collection Tubes) to avoid sedimentation into the columns.5. Centrifuge at 13,000 rpm for 15 seconds, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.6. Add 150 µl Buffer PB to the adsorption column and centrifuge at 13,000 rpm for 15 seconds.7. Add 400 µl Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first) and centrifuge at 13,000 rpm for 1 minute.8. Place the adsorbent column in a new centrifuge tube (self-provided), add 30-100 µl Buffer EB to the middle part of the adsorbent membrane, centrifuge at 13,000 rpm for 1 min, collect the plasmid DNA, and store at -20°C for long term storage.When the amount of extracted bacterial liquid is >600µl, the following procedure can be used:1. This kit can extract up to 3ml of bacterial solution, if the amount of extracted bacterial solution is more than 600µl, it is necessary to centrifuge the bacterial solution exceeding 600µl at 13,000rpm for 30 seconds (to collect the bacterial body), discard the supernatant and then add 600µl of bacterial solution, and then resuspend the bacterial body at the bottom of the tube thoroughly and then proceed to the following operation.2. Add 100µl Buffer L2 to the above centrifuge tube, gently invert the solution up and down 10 times, if the solution is not clarified, need to continue to invert the mixing until the solution becomes a clear purple color, the lysis time should not be more than 2 minutes. (If the solution is still turbid, the amount of bacteria is too large, and the amount of bacteria should be reduced appropriately.)3. Add 350 µl of Buffer N3 to the above centrifuge tube (please check that RNaseA has been added first) and immediately mix well by turning up and down until the purple solution turns completely yellow and a yellow precipitate is formed before proceeding to the next step. centrifuge at 13,000 rpm for 5 minutes.4. Transfer the supernatant to a new centrifuge tube, add 200 µl of isopropanol, mix up and down several times, mix well and transfer to the adsorbent column (Spin Columns DM with Collection Tubes), due to the amount of solution is too large, this time, it is necessary to centrifuge the column in two separate times, centrifugation at 13,000 rpm for 15 seconds, pour off the waste liquid in the collection tube, and put the adsorbent column back to the The adsorbent column should be placed back into the collection tube.5. Add 150 µl Buffer PB to the adsorption column and centrifuge at 13,000 rpm for 15 seconds.6. Add 400 µl Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first) and centrifuge at 13,000 rpm for 1 minute.7. Place the adsorbent column in a new centrifuge tube (self-provided), add 50-200 µl Buffer EB to the middle part of the adsorbent membrane, leave it at room temperature for 2 min, centrifuge at 13,000 rpm for 1 min, collect the plasmid DNA, and store it at -20°C for a long time... Read More |