| Description | Inquire | Alanine Aminotransferase (ALT), also known as serum glutamic-pyruvic transaminase (SGPT), is a pyridoxal-phosphate-dependent enzyme that catalyzes the reversible transfer of an amino group from alanine to α-ketoglutarate, generating pyruvate and glutamate. ALT is found primarily in liver and Alanine Aminotransferase (ALT), also known as serum glutamic-pyruvic transaminase (SGPT), is a pyridoxal-phosphate-dependent enzyme that catalyzes the reversible transfer of an amino group from alanine to α-ketoglutarate, generating pyruvate and glutamate. ALT is found primarily in liver and serum, but occurs in other tissues as well. Hepatocellular injury often results in an increase of serum ALT levels and serum ALT levels can be used as a marker for liver injury.ALT Activity Assay kit has been used to determine the activity of alanine aminotransferase (ALT) in serum samples... Read More | The bacterial viability / toxicity detection kit contains two fluorescent dyes. Nucgreen is a green nucleic acid dye that can stain live and dead bacteria; Ethd III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When nucgreen and ethd III are properly mixed, The bacterial viability / toxicity detection kit contains two fluorescent dyes. Nucgreen is a green nucleic acid dye that can stain live and dead bacteria; Ethd III is a red nucleic acid dye that only stains dead bacteria with damaged cell membranes. When nucgreen and ethd III are properly mixed, the bacteria with intact cell membrane appear green, while the bacteria with damaged cell membrane can appear green and red under different channels, respectively. A common criterion for bacterial viability is the ability to propagate in a suitable nutrient medium, known as a growth assay. This kit is generally in good agreement with the growth assay results in liquid or solid medium. However, under certain conditions, membrane damaged bacteria may recover and propagate in nutrient medium, and such bacteria will be identified as dead bacteria in this assay. On the contrary, some bacteria with intact membranes may not be able to propagate in nutrient medium, but will be recognized as viable bacteria in this assay. Therefore, if there is a large difference between the test results of this kit and the bacterial growth assay, the above possibilities should be considered. Component： Product parameters： NucGreen: Ex/Em = 503/530 nm (结合 DNA)；EthD-III: Ex/Em = 530/620 nm (结合 DNA)。Usage:1 Preparation of control samples for live and dead bacteria (optional)1. Cultivate 4 mL of bacteria in liquid medium until late logarithmic phase.2. Prepare two 1 mL bacterial solutions in an EP tube and centrifuge for 10-15 minutes under 5000-10000 g conditions.3. Remove the supernatant and add 0.3 mL of 0.85% NaCl resuspended bacteria to one of the EP tubes, and 1 mL of 0.85% NaCl resuspended bacteria to the other tube.4. Add 0.7 mL of isopropanol to a tube containing 0.3 mL of 0.85% NaCl, and mix thoroughly (with a final concentration of 70% isopropanol) to prepare a dead bacterial sample.5. Incubate the two samples at room temperature for 1 hour and mix every 15 minutes.6. Centrifuge the two samples at 5000-10000 g for 10-15 minutes.7. Remove the supernatant, add 1 mL of 0.85% NaCl to resuspend the bacteria in both samples, and centrifuge again as in step 6.8. Use a spectrophotometer to measure the absorbance values (OD670) of two bacterial suspensions at 670 nm.9. Adjust the density of the two bacterial suspensions (live and dead) to 108 bacteria/mL (OD670 ≈ 0.3), and then dilute with 0.85% NaCl at 1:100 to achieve a final density of 106 bacteria/mL.10. Mix two bacterial suspensions as shown in the table below to obtain the required live cell ratio: dead cell ratio.Table 1 Mix live and dead bacterial suspensions by a certain volume to achieve the required ratio of live and dead cellsLive cells: Dead cellsVolume of viable bacterial suspension（mL）Volume of dead bacterial suspension（mL）0:10001.010:900.10.920:800.20.830:700.30.750:500.50.5100:01.00II Staining methods for fluorescence microscopy observation1. Mix 1 volume of component A, NucGreen, and 2 volumes of component B, EthD-III, in a microcentrifuge tube. After thorough mixing, add 8 volumes of 0.85% NaCl solution to obtain a 100 x dye solution.2. Every 100 µ L bacterial suspension, add 1 µ 100 x dye solution of L.3. Mix thoroughly and incubate at room temperature in the dark for 15 minutes.4. Take 5 µ The bacterial suspension after L staining was dropped onto a glass slide with an 18 mm square cover glass.5. Observe under a fluorescence microscope. The fluorescence of live and dead bacteria can be observed simultaneously under any standard FITC long-acting filter. Alternatively, live (green fluorescent) and dead (red fluorescent) bacteria can be observed using FITC and Cy3 (or Texas Red) channels, respectively.Attention: (1) Before staining bacteria, attention must be paid to removing residues of growth media. Nucleic acid and other media components can bind to NucGreen and EthD-III dyes in some way, resulting in unacceptable staining changes. A simple washing step is usually sufficient to remove interfering media components from bacterial suspension. It is not recommended to use phosphate buffer solutions as they can reduce staining efficiency. (2) Before starting the formal experiment, the dye concentration should be adjusted to distinguish between NucGreen labeling live bacteria and EthD-III labeling dead bacteria. The optimal concentration may vary depending on the bacterial strain. It is generally best to use the lowest dye concentration that can provide sufficient signal. The above conditions have been optimized for staining live/dead cells of Escherichia coli.III Before starting the staining method experiment of flow cytometry, please read the precautions under the fluorescence microscope staining steps.According to Table 1, add 11 different proportions of live and dead bacteria to the EP tube. Each of the 11 samples has a volume of 1 mL.2. Add 12 µ The A component of L, NucGreen, and 24 µ The B component EthD-III of L was mixed in a microcentrifuge tube. Add 3 to each of the 11 samples µ Mix the mixed dyes of L thoroughly by blowing them up and down several times. (Note: Additional control bacterial samples need to be prepared for separate NucGreen and EthD-III staining)3. Incubate at room temperature in the dark for 15 minutes.4. Analyze each sample using a flow cytometer, detect NucGreen positive cells using FITC channels, and detect EthD-III positive cells using PI or PE channels.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. if the orifice plate is used for detection, a small amount of bacterial liquid can be left for imaging after standing for 10 min, which can effectively reduce the background. 3. in order to be closer to the real results, it is recommended to keep the brightness of red fluorescence consistent with that of green fluorescence in merge pictures. 4. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching. 5. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Staining of dead and live bacteria... Read More | Inquire | V669947 Component 50T Storage V669947A Buffer GL 15 mL RT V669947B Buffer GW1 (concentrate) 13 mL RT V669947C Buffer GW2 (concentrate) 15 mL RT V669947D Buffer RE 10 mL RT V669947E Proteinase K 12.5 mg RT V669947F Proteinase K Storage Buffer 1.25 mL RT V669947G Spin Columns RS with Collection Tubes V669947 Component 50T Storage V669947A Buffer GL 15 mL RT V669947B Buffer GW1 (concentrate) 13 mL RT V669947C Buffer GW2 (concentrate) 15 mL RT V669947D Buffer RE 10 mL RT V669947E Proteinase K 12.5 mg RT V669947F Proteinase K Storage Buffer 1.25 mL RT V669947G Spin Columns RS with Collection Tubes 50 RT V669947H RNase-Free Centrifuge Tubes (1.5 mL) 50 RTProductsThis kit is suitable for the extraction of viral RNA and DNA from fresh or frozen plasma, serum and cell-free body fluids. It is easy to operate as it does not require the use of organic solvents such as phenol and chloroform for extraction. The kit uses a unique buffer system to enable efficient and specific binding of viral nucleic acids in lysate to silica gel centrifugal adsorption columns. Inhibitors of PCR and enzyme reactions as well as residual impurities can be efficiently removed in a two-step effective rinsing step, and finally high purity viral nucleic acids can be obtained by using a low-salt buffer or water for elution. The purified viral nucleic acid is free of protein, nuclease and other impurities, and can be used directly in PCR, RT-PCR, Real-Time PCR, blotting experiments and so on.Self-contained reagent: anhydrous ethanol.Pre-experiment and Important Notes1. Add 1.25ml Proteinase K Storage Buffer to Proteinase K to dissolve it and store it at -20℃. Do not leave the prepared Proteinase K at room temperature for a long time, and avoid repeated freezing and thawing to avoid affecting its activity. Do not add Proteinase K directly into Buffer GL.2. Repeated freezing and thawing of the sample should be avoided, as this may result in smaller DNA fragments and a decrease in the amount of extracted DNA.3. Avoid repeated freezing and thawing of serum or plasma, which can lead to protein denaturation or precipitation, reducing the viral titer and thus affecting the yield of extracted viral nucleic acids.4. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the label instructions of the reagent bottle before first use.5. Check Buffer GL for crystallization or precipitation before use. If crystallization or precipitation occurs, redissolve Buffer GL in a water bath at 56℃.Procedure1. Take a 1.5 ml centrifuge tube (self-provided) and add 20 µl Proteinase K.2. Add 200 µl serum or plasma to the centrifuge tube. Add 200µl Buffer GL and vortex and shake for 15 seconds.Note: 1) Sample volume less than 200 µl can be made up by adding 0.9% NaCl (self-provided). 2) In order to ensure effective lysis of the sample, the sample needs to be mixed well with Buffer GL after adding Buffer GL.3. Incubate at 56°C for 15 minutes, centrifuge briefly, and collect the solution from the wall of the tube to the bottom of the tube.4. 250 µl of anhydrous ethanol was added, vortexed and shaken for 15 seconds, left at room temperature for 5 minutes, centrifuged briefly, and the solution on the wall of the tube was collected at the bottom of the tube.Note: If the ambient temperature exceeds 25°C, anhydrous ethanol should be used after pre-cooling on ice.5. Add the solution obtained in step 4 to the adsorbent column (RNase-Free Columns RS) that has been loaded into the collection tube, and if the solution cannot be added at one time, it can be transferred in several times. centrifuge the column at 12,000 rpm (~13,400 × g) for 1 min, pour off the waste liquid in the collection tube, and put the column back into the collection tube.6. Add 500 µl of Buffer GW1 to the adsorption 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 adsorption 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 at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the adsorption column back into the collection tube.Note: Step 7 can be repeated if further DNA purity is required.8. Add 500 µl of anhydrous ethanol to the adsorbent column and 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.9. Centrifuge at 12,000 rpm for 3 minutes 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 the removal of residual ethanol from the adsorbent column; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).10. Place the adsorption column in a new collection tube (RNase-Free Centrifuge Tube), add 20-150 µl of Buffer RE or sterilized water overhanging the middle of the adsorption column membrane, leave it at room temperature for 2-5 minutes, and then centrifuge it at 12,000 rpm for 1 minute to collect the nucleic acid solution.Note: 1) If the downstream experiment is sensitive to pH or EDTA, you can use sterilized water for elution. The pH of the eluent has a great influence on the elution efficiency, if water is used as the eluent it should be ensured that its pH is 7.0-8.5 (the pH of water can be adjusted to this range with NaOH), and the elution efficiency is not high when the pH is lower than 7.0.(2) For long-term storage, please store the DNA solution at -20℃ and the RNA solution at -70℃.3) If the final concentration of DNA/RNA is to be increased, the DNA/RNA eluate obtained in step 10 can be re-spiked onto the adsorbent membrane and step 10 repeated... Read More |