| Description | Triglycerides (TG), also known as triacylglycerols, are fat molecules formed from three long-chain fatty acids and glycerol. They are the most abundant lipids in the human body. Most tissues can utilize the breakdown products of triglycerides for energy. Simultaneously, tissues like the liver and Triglycerides (TG), also known as triacylglycerols, are fat molecules formed from three long-chain fatty acids and glycerol. They are the most abundant lipids in the human body. Most tissues can utilize the breakdown products of triglycerides for energy. Simultaneously, tissues like the liver and adipose tissue can synthesize triglycerides. The enzymatic method for measuring TG is commonly used in biochemical assays due to its characteristics: 1. High sensitivity, accuracy, and precision; 2. Use of mild reaction conditions; 3. Simple operation; 4. Suitable for semi-automatic biochemical analyzers.Detection Principle: Triglycerides are hydrolyzed by lipoprotein lipase (LPL) into glycerol and free fatty acids. Glycerol is then phosphorylated by glycerol kinase (GK) and adenosine triphosphate (ATP) to form glycerol-3-phosphate (G-3-P). G-3-P is subsequently oxidized by glycerol-3-phosphate oxidase (GPO), producing hydrogen peroxide. The hydrogen peroxide, in the presence of peroxidase (POD), 4-aminoantipyrine (4-AAP), and phenol (collectively known as PAP), reacts to form a red-colored quinoneimine dye (Trinder reaction). The quinoneimine dye has a maximum absorption at 510 nm. The absorbance is directly proportional to the triglyceride concentration in the sample and can be measured using a microplate reader between 500-520 nm.This kit is used for the quantitative determination of triglyceride content in serum, cells, tissues, and other samples from humans or animals. This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.Component100TStorageBuffer Solution24 mL2-8℃. Store in the dark.Enzyme Reagent6 mL2-8℃. Store in the dark.Glycerol Standard (1.7 mmol/L)1 mL2-8℃User-Prepared Instruments and ReagentsddH₂O, Physiological Saline or PBSCentrifuge tubes or small test tubes, Water bath or incubatorMicroplate reader, 96-well plate, Semi-automatic biochemical analyzerExperimental Procedure1. Sample Preparation1.1 Serum, Plasma, Cerebrospinal Fluid SamplesSerum or plasma separated from the test sample should not be hemolyzed. Assay directly. If the concentration exceeds the linear range, dilute with physiological saline before assaying.1.2 Cell Samples(1) Take an appropriate amount of cells (generally recommended >10⁶), centrifuge at 1000 g for 10 min, discard the supernatant, keep the pellet.(2) Wash the pellet 1-2 times with PBS or physiological saline, centrifuge at 1000 g for 10 min, discard the supernatant, keep the pellet.(3) Add 200-300 µL of PBS or physiological saline to homogenize. Sonicate the cells on ice (power 300W, pulse 3-5s, interval 30s, repeat 3-5 times). Alternatively, homogenize manually. Do not centrifuge the prepared homogenate. Alternatively, lyse with 1-2% Triton X-100 on ice for 30-60 min. Do not centrifuge the prepared lysate.1.3 Tissue SamplesAccurately weigh an appropriate amount of tissue sample. Add physiological saline or PBS at a mass (g) to volume (mL) ratio of 1:9. Homogenize manually or mechanically on ice. Centrifuge at 2500-3000 g for 10 min. Collect the supernatant for assay.2. Preparation of GPO-PAP Working SolutionBefore use, mix the Buffer Solution and Enzyme Reagent at a 4:1 volume ratio. Mix well. Store at 4°C.3. TG Assay Steps using Microplate Reader3.1 Add reagents sequentially to the 96-well plate according to the table below. Mix thoroughly and incubate at 37°C in a water bath or incubator for 10 minutes.Reagent (µL)Blank WellStandard WellTest WellddH2O2.5//Glycerol Standard (1.7 mmol/L)/2.5/Test Sample//2.5GPO-PAP Working Solution2502502503.2 Measure the absorbance between 500-520 nm using the microplate reader. Zero the instrument with the blank well, then read the absorbance of the standard well and all test wells.4. TG Assay Steps using Semi-Automatic Biochemical Analyzer4.1 Instrument Parameter Settings:WavelengthTemperatureDelay TimeMeasurement TimeReagent BlankReaction TypeAspiration Volume510-550nm37℃2s2sYesEndpoint800µL4.2 Add reagents sequentially to tubes according to the table below. Mix thoroughly and incubate at 37°C in a water bath for 10 minutes.Reagent (µL)Blank TubeStandard TubeTest TubeddH2O10//Glycerol Standard (1.7 mmol/L)/10/Test Sample//10GPO-PAP Working Solution1000100010004.3 Zero the instrument with the blank tube, then read the absorbance of the standard tube and all test tubes.5. Calculation Formula5.1 For serum, plasma, and other liquid samples (Blank zeroed):TG (mmol/L) = (Absorbance of Test Well/Tube / Absorbance of Standard Well/Tube) × 1.7 mmol/L5.2 For cell, tissue, and other samples (Blank zeroed):TG (mmol/g prot) = (Absorbance of Test Well/Tube / Absorbance of Standard Well/Tube) × 1.7 mmol/L / Sample Protein Concentration (mg/mL)Reference Interval (Healthy Adults)Desirable range: < 1.7 mmol/L (< 150 mg/dL)Borderline high: 1.7 – 2.25 mmol/L (150 – 199 mg/dL)High: 2.26 – 5.64 mmol/L (200 – 499 mg/dL)Very high: ≥ 5.65 mmol/L (≥ 500 mg/dL)Precautions1. Avoid repeated freeze-thaw cycles for the low-temperature reagents mentioned above to prevent inactivation or decreased efficiency.2. The GPO-PAP Working Solution should be prepared immediately before use and is not suitable for long-term storage at 4°C.3. This method can be directly used to detect TG content in cerebrospinal fluid but cannot directly detect TG in urine, as untreated urine contains reducing substances that interfere with the peroxidase reaction.4. If test samples cannot be assayed immediately, they should be stored at 2-8°C and are stable for 3 days.5. The linear range of this method is up to 9.0 mmol/L. If the sample TG concentration is too high, results may be falsely low. Dilute the sample with physiological saline and re-assay, multiplying the result by the dilution factor.6. The working reagent should be protected from contamination by substances like glucose and cholesterol.7. The reagent is susceptible to oxidation by air, turning red. A blank measurement is necessary.8. For your safety and health, please wear a lab coat and disposable gloves during operation.9.Use the reagents as soon as possible after opening to prevent affecting subsequent experimental results... Read More | Inquire | 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 content R669871Component50 TStorageR669871ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669871B10×Reaction Buffer1mL-20℃. Avoid freeze/thaw cycle. R669871CBuffer DS30 mLRTR669871DBuffer GTL15 mLRTR669871EBuffer GL25 mLRTR669871FProteinase K12.5 mgRTR669871GProteinase K Product content R669871Component50 TStorageR669871ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669871B10×Reaction Buffer1mL-20℃. Avoid freeze/thaw cycle. R669871CBuffer DS30 mLRTR669871DBuffer GTL15 mLRTR669871EBuffer GL25 mLRTR669871FProteinase K12.5 mgRTR669871GProteinase K Storage Buffer1.25 mLRTR669871HBuffer RW140 mLRTR669871IBuffer RW2 (concentrate)11 mLRTR669871JRNase-Free Water10 mLRTR669871KSpin Columns RS with Collection Tubes50 setsRTR669871LRNase-Free Centrifuge Tubes (1.5 mL)50 EART Product IntroductionThis kit is suitable for effectively purifying total RNA from formalin fixed and paraffin embedded tissues. Suitable for extracting total RNA with improved purity from paraffin embedded tissues or sections less than 30mg. This kit does not require the use of phenol/chloroform extraction or isopropanol precipitation, and can complete the extraction of multiple samples within one hour. This product uses specially optimized lysis solution and protease K to release RNA from formalin fixed or tissue slice samples without overnight operation; After digestion, the sample is incubated at a higher temperature to remove the inhibitory effect caused by formalin cross-linking, effectively releasing RNA from tissue slices and avoiding endangering RNA integrity; The optimized buffer system allows RNA in the lysis solution to specifically bind to the silica gel adsorption membrane, while other pollutants can flow through the membrane; It can be effectively removed through rinsing steps, and the washed RNA can be directly used for experiments such as RT-PCR, Real Time PCR, and Western blot analysis.Self prepared reagents: anhydrous ethanol (newly opened or dedicated for RNA extraction), 10mM PBS (pH 7.4).Preparation and important precautions before the experiment1. Add 0.625ml Protein K Storage Buffer to Protein K to dissolve it and store at -20 ℃. The prepared Protein K should not be left at room temperature for a long time to avoid repeated freeze-thaw cycles, which may affect its activity.2. To prevent RNase pollution, attention should be paid to the following aspects:1) Use RNase free plastic products and gun heads to avoid cross contamination.2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3) Prepare the solution using water without RNase.4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.3. After obtaining the sample, it should be fixed in 4% -10% formalin as soon as possible, with a suitable fixation time of 14-24 hours. Excessive time can lead to RNA breakage and affect downstream experiments.4. Ensure that the sample before embedding is thoroughly dehydrated, as residual formalin will inhibit the action of Protein K.5. Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.Before use, please check if there is any crystallization or precipitation in Buffer GTL, Buffer GL, and Buffer DS. If there is any crystallization or precipitation, please dissolve Buffer GTL, Buffer GL, and Buffer DS again in a 56 ℃ water bath.Operation steps1. Sample processing1a. Paraffin embedded sample: Use a surgical knife to trim off excess paraffin from the tissue block, expose the tissue, and cut into 5-10 µ m thin slices.Attention: If the surface of the sample has already been exposed to air, please discard 2-3 pieces that come into contact with the air and do not use them.1b. Samples in fixed solutions such as formalin: Take approximately 20mg of the sample, cut it into small pieces, place it in a centrifuge tube, and add 500 µ 10mM PBS (PH7.4), vortex oscillation, centrifugation at 12000 rpm (~13400 × g) for 1 minute, discard the supernatant, repeat 3 times, and proceed directly to step 3.2. Choose option A or option B to remove paraffinOption AA1. Take approximately 1 × 1cm2 of slices (4-5 slices in total) and place them in a centrifuge tube (prepared by oneself), then add 500 slices µ L Buffer DS, vortex oscillation for 10 seconds. Incubate at 56 ° C for 3 minutes.Centrifuge at A2.12000 rpm for 2 minutes, be careful to discard the supernatant and avoid attracting sediment.Option BB1. Take approximately 4-5 slices of approximately 1 × 1 cm2 and place them in a centrifuge tube (self prepared). Add 1ml of xylene, cover the tube tightly, and vortex for 10 seconds.B2.Centrifuge at 12000 rpm for 2 minutes, be careful to remove the supernatant and avoid removing sediment.B3. Add 1ml of anhydrous ethanol, vortex and shake well. Centrifuge at 12000 rpm for 2 minutes, discard the supernatant, and be careful not to absorb or discard the sediment.B4. Open the tube cover and incubate at room temperature or up to 37 ° C for 10 minutes until there is no ethanol residue.3. Add 150µ L Buffer GTL, resuspended precipitation; Join 10µl Protein K, vortex oscillation mixing.4.Incubate at 56 ℃ for 15 minutes until the sample is completely dissolved. Incubate at 80 ℃ for 15 minutes. Short centrifugation allows the solution on the tube wall to be collected to the bottom of the tube.Note: 1) The purpose of this step is to repair nucleic acids denatured by formaldehyde. Incubating at a high temperature or for too long may cause RNA breakage, resulting in RNA fragments.2) The sample incubated at 56 ℃ can be placed at room temperature until the temperature of the water or dry bath reaches 80 ℃, and then the sample can be incubated at 80 ℃.5. Place on ice for 3 minutes, centrifuge at 12000 rpm for 15 minutes, transfer the supernatant to a new centrifuge tube, be careful not to suck sediment.6. Add 320 to the supernatant µ L Buffer GL, vortex oscillation thoroughly mixed.7. Join 720 µ Mix anhydrous ethanol thoroughly with vortex oscillation.Attention: After adding anhydrous ethanol, there may be a small amount of precipitate precipitation, but it does not affect subsequent operations.8. Add all the solutions obtained in step 7 to the spin columns RS that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. Centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.Optional steps: If genomic DNA needs to be removed, the following steps can be followeda. Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and place the adsorption column back into the recovery manifold.b. Preparation of DNase I mixture: Take 52 µ Add 8 RNase Free Water to it µ 10 x Reaction Buffer and 20 µ DNase I (1U/ µ l) Mix well and prepare to a final volume of 80 µ The reaction solution of L.c. Add 80 µ l of DNase I mixture directly to the adsorption column and incubate at 20-30 ℃ for 15 minutes.d. Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and place the adsorption column back into the recovery manifold.9. Add 500 to the adsorption column µ Buffer RW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.10. Repeat step 9.Centrifuge at 11.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which will affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).12. Place the adsorption column in a new RNase free centrifuge tube, and add 20-50µl to the middle of the adsorption column in the air Place RNase Free Water at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store RNA at -20 ℃.Note: 1) The volume of RNase Free Water should not be less than 20 µ l. Small volume affects the recovery rate. 2) If you want to increase RNA production, you can use 20-50 µ Repeat step 12 for the new RNase Free Water.3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column and repeat step 12... Read More |