| Description | Biotin Ligase (also known as BirA, EC 6.3.4.15) can activate biotin to form biotinyl-5'-adenylate, and specifically transfer biotin to biotin receptor proteins (such as AviTag fusion proteins), thereby biotinylating the proteins.Component ListB1505408Component20µg2×20µBiotin Ligase (also known as BirA, EC 6.3.4.15) can activate biotin to form biotinyl-5'-adenylate, and specifically transfer biotin to biotin receptor proteins (such as AviTag fusion proteins), thereby biotinylating the proteins.Component ListB1505408Component20µg2×20µgStorageB1505408ABiotin-Protein Ligase (1mg/ml)20µl2×20µl-80℃. Avoid freeze/thaw cycle.B1505408B10×Biotin Ligase Buffer A500µl1 mL-80℃. Avoid freeze/thaw cycle.B1505408C10×Biotin Ligase Buffer B500µl1 mL-80℃. Avoid freeze/thaw cycle.Activity and Purity The biological activity of Biotin-Protein Ligase is ≥ 7,500 Units/µg. Purity is > 95%, with no detectable protease activity.Storage Conditions1. Shipped on dry ice. After thawing, aliquot the product and avoid repeated freeze-thaw cycles.2. For long-term storage of Biotin-Protein Ligase, store at -80°C; for short-term use, store at -20°C; for frequent use, it can be stored at 4°C for a short period. When stored at 4°C, the enzyme can retain > 90% of its activity for three months.3. Store Ligase Buffer A and Ligase Buffer B at -20°C; repeated freeze-thaw cycles are acceptable.Example of Reaction SystemReactant ComponentVolume10×Biotin Ligase Buffer A2.5 µl10×Biotin Ligase Buffer B2.5 µlBiotin Ligase0.17 µlProtein (polypeptide) substrate1 - 5 µgdH₂Oup to 25 µl Total volume is 25 µl. Incubate at 25°C for 12 - 16 hours to fully biotinylate the substrate.Precautions1. The substrate of this product is AviTag fusion protein.2. If the substrate protein (polypeptide) needs to be stored at a lower temperature, the reaction temperature can be appropriately reduced and the enzymatic reaction time should be prolonged.3. Many common buffer components such as sodium chloride (>100 mM), glycerol (>5% W/V), and ammonium sulfate (>50 mM) can inhibit the activity of biotin ligase. When these reagents are necessary for the substrate protein (polypeptide), their concentrations should be reduced as much as possible.4. The substrate can contain Tris (pH 8.0), with an optimal concentration of 10 mM and no more than 50 mM.FAQsQ: How to prevent proteases from degrading the substrate?A: The biotin ligase of this product undergoes strict quality control and has no protease activity. If the substrate protein used for biotinylation is unpurified crude protein, it is recommended to add an appropriate amount of protease inhibitors to prevent degradation during the biotinylation process.Q: How to determine the optimal amount of this enzyme and reaction conditions for the reaction?A: Due to the great differences in the properties of different proteins and the certain uncertainty in biological enzyme reactions, the reaction conditions described in the manual are not completely applicable to all proteins. It is recommended that users conduct a pre-experiment: first take a small amount of protein, divide it into several equal amounts of substrates, add enzymes with different dilutions, corresponding amounts of Buffer A and B, react at 30°C for 1 h (or other time and temperature conditions), terminate the reaction with SDS-PAGE Loading Buffer, perform electrophoresis and transfer to an NC membrane for Western Blotting detection (after blocking with BSA or skim milk powder, directly incubate with commercially available HRP-Streptavidin, and develop color with DAB), compare the biotinylation situation, and select the optimal conditions. Since the binding force between Streptavidin and Biotin is extremely strong, only a small amount of biotinylated protein is needed for observation results when used for Western Blotting detection.Q: What should I do if the concentration of my substrate protein is quite low and it is not easy to concentrate during biotinylation?A: Add more enzyme and appropriately prolong the reaction time when conducting the enzymatic reaction with the same amount of substrate. However, there is a problem that cannot be ignored: the enzyme is a protein, and when the amount of enzyme increases, the amount of protein introduced into the system also increases. If subsequent experiments do not allow the presence of a large amount of heterologous proteins, then prolonging the reaction time will be the only choice.Q: Does detergent have any effect on the biotinylation reaction?A: Through experimental detection, 0.2% Tween 20 has no effect on the reaction. This product is only for experimental scientific research use. If any unit or individual uses this product for other special purposes specifically stipulated by the state such as clinical diagnosis and treatment, our company will not bear any responsibility... Read More | Format:2-ComponentEnzyme:Horseradish peroxidase | Product introduction：Reporter gene detection is an important tool for analyzing the interaction between potential cis elements (such as promoters, enhancers and silencers) and trans acting factors in the flanking region of structural genes in the field of modern molecular biology. Firefly Product introduction：Reporter gene detection is an important tool for analyzing the interaction between potential cis elements (such as promoters, enhancers and silencers) and trans acting factors in the flanking region of structural genes in the field of modern molecular biology. Firefly luciferase is widely used in gene regulation and drug screening. Firefly luciferase is a protein with a molecular weight of about 61 KD. 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. The optical signal of this kit is a kind of instantaneous light, which needs to be detected immediately after adding the working solution. The half-life of optical signal is about 5 min.Instruction：1.Working fluid configuration ( 1 ) Restore all components to room temperature. ( 2 ) The component B ( stock solution ) was fully diluted with component A to prepare a 0.2 mg / mL firefly luciferase working solution, which was vortexed and shaken to ensure full mixing. Note : The firefly luciferase working solution cannot be repeatedly frozen and thawed. If the dosage of a single experiment is small, it is recommended to subpackage according to a single dosage. At room temperature, the activity decreased by about 10 % after the working solution was configured for 3 h, and the activity decreased by about 25 % after 5 h. 2.chemiluminescence value detection ( 1 ) The cell culture plate was taken out from the incubator and incubated at room temperature for 20 min to restore it to room temperature ( 22-25 ° C ). ( 2 ) Add the same volume of firefly luciferase working solution with the medium to the culture plate and mix well. ( 3 ) Incubation at room temperature for 5 min. Note : The incubation time can be adjusted according to cell type and cell number. ( 4 ) The values were read by multifunctional microplate reader or chemiluminescence instrument ( instrument parameters : the determination time was 10 s, the determination interval was 2 s ).Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. the strongest wavelength of bioluminescence catalyzed by firefly luciferase is 560 nm. 3. to prevent interference between holes, it is recommended to use white opaque orifice plate.Recommendation：Component B is recommended to use sterile water in advance to configure 2 mg / mL storage solution, A component and B component configured as storage solution, and small batch packaging according to the experimental requirements. The detection working fluid is recommended to be used now to avoid repeated freezing and thawing. Component：One-Step Firefly Luciferase Assay Buffer；D-Luciferin Scope of application：Mainly used for ADCC detection... 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 | R669988 Component 50T Storage R669988A DNase I 1000 U -20℃. Avoid freeze/thaw cycle. R669988B 10×Reaction Buffer 1000 µL -20℃. Avoid freeze/thaw cycle. R669988C Buffer RL 35 mL RT R669988D Buffer RLC 35 mL RT R669988E Buffer RW1 40 mL RT R669988F Buffer RW2 (concentrate) 11 mL R669988 Component 50T Storage R669988A DNase I 1000 U -20℃. Avoid freeze/thaw cycle. R669988B 10×Reaction Buffer 1000 µL -20℃. Avoid freeze/thaw cycle. R669988C Buffer RL 35 mL RT R669988D Buffer RLC 35 mL RT R669988E Buffer RW1 40 mL RT R669988F Buffer RW2 (concentrate) 11 mL RT R669988G RNase-Free Water 10 mL RT R669988H Spin Columns FL with Collection Tubes 50 sets RT R669988I Spin Columns RM with Collection Tubes 50 sets RT R669988J RNase-Free Centrifuge Tubes (1.5 mL) 50 EA RTProductsThis kit is used for the extraction and purification of high-quality total RNA from a variety of plants, and is also suitable for the extraction of fungal mycelial RNA. The unique separation column is used for homogenization and filtration of high viscosity plant or fungal lysates, while the silicon matrix membrane is used to adsorb the RNA for purification, so that various contaminants, such as polysaccharides, are effectively removed by washing, and the eluted RNA can be directly used in various downstream experiments. The molecular weight of RNA extracted by this kit is more than 200 bases, with high purity and almost no DNA residue. For RNA experiments that are very sensitive to trace DNA, the residual DNA can be removed by digestion on a column using RNase-free DNase. The extracted RNA can be used in Northern Blot, Dot Blot, RT-PCR and in vitro translation experiments.Self-contained reagents: β-mercaptoethanol, anhydrous ethanol (freshly opened or for RNA extraction).Pre-experiment Preparation and Important Notes1. To prevent RNase contamination, attention should be paid to the following aspects:1) Use RNase-free plastics and tips to avoid cross-contamination.2) RNase-free water should be used to prepare the solution.(3) Operators wear disposable masks and gloves, and change gloves diligently during the experiment.2. To prevent RNase contamination, attention should be paid to the following aspects:1) Use RNase-free plastics and tips to avoid cross-contamination.(2) Glassware should be dry-roasted at 180°C for 4 hours before use, and plasticware can be soaked in 0.5M NaOH for 10 minutes, rinsed thoroughly with water and autoclaved.3) RNase-free water should be used to prepare the solution.(4) Operators wear disposable masks and gloves, and change gloves diligently during the experiment.3. Avoid repeated freezing and thawing of the extracted samples, otherwise it will affect the amount and quality of RNA extraction.4. Please add β-mercaptoethanol to Buffer RL before use, add 10µl of β-mercaptoethanol to 1ml of Buffer RL, it can be stored for 1 month at room temperature. Buffer RL with β-mercaptoethanol can be stored at room temperature for 1 month. β-mercaptoethanol is not required for use of Buffer RLC.5. Anhydrous ethanol should be added to Buffer RW2 before first use according to the instructions on the reagent bottle label.6. If precipitation occurs in Buffer RL and Buffer RLC, heat to dissolve and leave at room temperature.7. All centrifugation steps are carried out at room temperature and all steps are performed quickly. Procedure1. 50-100 mg of plant tissue is quickly ground to a powder in liquid nitrogen and added to 600 µl of Buffer RL (check for addition of β-mercaptoethanol before use) or Buffer RLC. vortexing and oscillating to allow for adequate lysis.Note: 1) The main component of Buffer RL is guanidine isothiocyanate, which is suitable for lysis of most plant tissues. However, in some plant tissues (e.g. endosperm of corn), due to the special secondary metabolites, guanidine isothiocyanate causes precipitation of the sample, resulting in poor RNA extraction, in this case, Buffer RLC can be added instead of Buffer RL.2) Incubation at 56°C for 1-3 minutes helps tissue lysis, but do not incubate at high temperatures for plants with high starch content.2. Transfer all the liquid obtained in step 1 to an adsorption column (Spin Columns FL) that has been loaded into a collection tube, centrifuge at 12,000 rpm (~13,400 x g) for 2 minutes, and transfer the supernatant from the collection tube to a new centrifuge tube (supplied).Note: 1) The tip of the tip of the gun can be cut off when aspirating liquids to facilitate sampling.2) Spin Columns FL removes most of the debris, but a small portion will still flow out and a precipitate will form in the collection tube after centrifugation, so be careful to avoid aspirating the precipitate when proceeding to the next step.3. Add 0.5 times the volume of anhydrous ethanol to the clean lysate obtained in step 2 and mix rapidly.Note: Precipitation may occur upon addition of ethanol, but does not affect subsequent tests.4. Transfer the solution obtained in the previous step to the Spin Columns RM in the collection tube. If it is not possible to add all of the solution to the column at one time, centrifuge the column at 12,000 rpm for 15 seconds in two batches, discard the waste solution and put the column back into the collection tube.5. Add 350 µl Buffer RW1 to the adsorbent column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid and put the adsorbent column back into the collection tube.6. Preparation of DNase I mixture: Take 52µl of RNase-Free Water, add 8µl of 10×Reaction Buffer and 20µl of DNase I (1U/µl) to it, mix well, and make a final volume of 80µl of reaction solution.7. Add 80µl of DNase I mixture directly to the adsorption column and incubate at 20-30°C for 15 minutes.8. Add 350 µl of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid and put the column back into the collection tube.9. Add 500 µl of Buffer RW2 to the column (check that anhydrous ethanol is added before use), centrifuge at 12,000 rpm for 15 seconds, and discard the waste solution.10. Repeat step 9.11. Place the adsorbent column back into the collection tube, centrifuge at 12,000 rpm for 1 minute, and allow the column to come to room temperature for a few minutes to thoroughly dry out the anhydrous ethanol in the adsorbent column.Note: The purpose of this step is to remove residual ethanol from the adsorption column; ethanol residue can interfere with subsequent enzymatic reactions (zymography, PCR, etc.).12. Load the adsorption column into a new centrifuge tube, add 30-50 µl of RNase-Free Water to the middle of the adsorbent membrane, leave it at room temperature for 1 minute, centrifuge at 12,000 rpm for 1 minute, and store the resulting RNA solution at -70°C to prevent degradation.Note: 1) The volume of RNase-Free Water should not be less than 30 µl, too small volume affects the recovery rate.2) If you want to increase the RNA yield, repeat step 12 with 30-50 µl of fresh RNase-Free Water.3) If the RNA concentration is to be increased, the resulting solution can be reintroduced into the adsorption column and step 12 repeated... Read More |