| Description | Inquire | The Endo F Multi-Kit will deglycosylate N-linked glycans in both native and denatured conditions. Each enzyme has a distinct specificity for N-linked glycan release. One can choose to use the three enzymes in combination to completely remove all N-linked glycans present on a glycoprotein or peptide,The Endo F Multi-Kit will deglycosylate N-linked glycans in both native and denatured conditions. Each enzyme has a distinct specificity for N-linked glycan release. One can choose to use the three enzymes in combination to completely remove all N-linked glycans present on a glycoprotein or peptide, or to use each enzyme independently and thereby determine the type of N-glycans present.Product DescriptionThe Endo F Multi-kit is recommended to deglycosylate native proteins that are resistant to PNGase F cleavage under non-denatured conditions due to the glycan location within the protein’s three-dimensional structure, as these enzymes are known to be less sensitive to protein conformation.Each of the enzymes has a different N-linked glycan specificity:Endoglycosidase F1 cleaves high mannose and some hybrid type N-glycansEndoglycosidase F2 releases biantennary and high mannose glycans (at a 40X reduced rate)Endoglycosidase F3 will release triantennarry and fucosylated biantennary N-glycansContents1 vial: Endo F1- 20 µl (0.3 U)20 mM Tris-HCl pH 7.51 vial: Endo F2- 20 µl (0.1 U)10 mM sodium acetate, 25 mM NaCl, pH 4.51 vial: Endo F3- 20 µl (0.1 U)20 mM Tris-HCl pH 7.51 vial: 5x Reaction Buffer - 400 µl250 mM sodium acetate, pH4.51 vial: 5x Reaction Buffer - 400 µl250 mM sodium phosphate, pH5.5Specific ActivityDefined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micro-mole of denatured Ribonuclease B (Endo F1) or porcine fibrinogen peptides (Endo F2/F3) in 1 minute at 37°C, pH 5.5 (PH 4.5 for Endo F3). Cleavage is monitored by SDS-PAGE.FormulationThe enzymes are provided as a sterile-filtered solution.StabilitySeveral days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly.SpecificityEndo F1 cleaves Asparagine-linked (N-linked) high mannose or hybrid oligosaccharides. Endo F2 cleaves N-linked biantennary oligosaccharides and high mannose (at a 40X reduced rate). Endo F3 cleaves free or N-linked fucosylated biantennary or triantennary oligosaccharides,as well as triamannosylchitobiose core structures. These enzymes cleave between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. The recombinant version is not glycosylated, which may result in properties differing from the native protein.Quality & PurityEndo F1, Endo F2, and Endo F3 are tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37°C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The absence of exoglycosidase contaminants is confirmed by extended incubations with the corresponding pNP-glycosides. Directions for use 1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 34 µl final volume with de-ionized water. 2. Add 10 µl Endo F2 &F3 5x Reaction Buffer, 250 mM sodium acetate pH 4.5. Use Endo F1 buffer, 250 mM sodium phosphate pH 5.5 if you are using the Endo F1 enzyme alone. 4. Add 2.0 µl of each enzyme to the reaction. Incubate 3 hours at 37°C. Monitor cleavage by SDS-PAGE. Applications– Deglycosylation of native proteins resistant to PNGase F cleavage– Determination of glycan type (high mannose, biantennary, tri/tetrantennary)– Deglycosylating proteins which normally precipitate when deglycosylating– X-Ray CrystallographyThese three enzymes cleave asparagine-linked (N-linked) oligosaccharides between the two GlcNAc residues in the core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine, enhancing the solubility of the protein. In contrast, PNGase F removes the oligosaccharide intact... Read More | Product content: G665990Component200 TStorageG665990ABuffer PG100 mLRTG665990BBuffer PS60 mLRTG665990CBuffer PW (concentrate)50 mLRTG665990DBuffer EB30 mLRTG665990ESpin Columns DM with Collection Tubes200 EART Product Introduction:This kit uses a new silicon-based plasma membrane technology and Product content: G665990Component200 TStorageG665990ABuffer PG100 mLRTG665990BBuffer PS60 mLRTG665990CBuffer PW (concentrate)50 mLRTG665990DBuffer EB30 mLRTG665990ESpin Columns DM with Collection Tubes200 EART Product Introduction:This kit uses a new silicon-based plasma membrane technology and reagent formulation. Through the unique centrifugal adsorption column and the DNA washing elution step, 100 bp-10 kb DNA fragments can be recovered and purified from ordinary or low melting point agarose gel. The sol speed is fast and the recovery rate is high. The sol solution contains a pH indicator, which can be used to determine whether the sol recovery has reached the optimal state based on its color. Each adsorption column can adsorb up to 10 µ G DNA, while effectively removing impurities such as primers, enzymes, mineral oil, and agarose. The purified and recovered DNA has high purity and concentration, good integrity, and can be directly used for molecular biology experiments such as sequencing, linking and transformation, labeling, and in vitro transcription.Self prepared reagents: anhydrous ethanol, isopropanol.Preparation and important precautions before the experiment:1.Before the first use, anhydrous ethanol should be added to the Buffer PW according to the instructions on the reagent bottle label.2. Before use, please check the Buffer PG. If crystallization or precipitation occurs, it can be left in a 37 ℃ water bath for 3-5 minutes to restore clarity.3. It is best to use a new electrophoresis buffer during electrophoresis to avoid affecting the electrophoresis and recovery efficiency; The following experiment requires high requirements, please use TAE electrophoresis buffer as much as possible.4.When cutting glue, the UV irradiation time should be as short as possible to avoid damage to DNA.5. The recovery rate is related to the initial amount of DNA and the elution volume. The smaller the initial amount, the smaller the elution volume, and the lower the recovery rate.6. Preheat the water bath to 50 ℃.7. Buffer PG contains a pH indicator. When the pH is ≤ 7.5, the color of the solution is yellow, and DNA can effectively bind to the membrane. When the pH is too high, the color of the solution turns orange red and purple, which needs to be adjusted.8. All centrifugation steps can be performed at room temperature.Operation steps:1. Cut the single purpose DNA strip from the agarose gel (try to cut the excess), put it into a clean centrifuge tube (self prepared), and weigh and calculate the weight of the gel (record the weight of the centrifuge tube in advance).Attention: If the volume of the adhesive block is too large, it can be cut into small pieces.2. Add one time of the volume of Buffer PG (if the gel weighs 100 mg, its volume can be regarded as 100 µ l. And so on.3.50 ℃ water bath and gently invert the centrifuge tube every 2-3 minutes until the sol turns yellow to ensure full dissolution of the gel block. If there are still unsolved glue blocks, you can add some more sol solution or continue to let it stand for a few minutes until the glue blocks are completely dissolved.Note: 1) After the gel is completely dissolved, the gel solution is yellow, and subsequent operations can be carried out; If the glue solution is orange red or purple, 10-30 can be added to the glue solution µ 3 M sodium acetate (pH 5.0), adjust the color of the solution to yellow before proceeding with subsequent operations.2) After the gel block is completely dissolved, it is best to lower the temperature of the gel solution to room temperature before loading the column. The adsorption column has a weaker ability to bind DNA at higher temperatures.4. (Optional step) When the recovered fragment is less than 300 bp, add 1/2 of the gel volume of isopropanol, and mix it upside down (if the gel weighs 100 mg, add 50 µ Isopropanol of L.5. Column balance: Add 200 to the spin columns DM that have been loaded into the collection tube µ Centrifuge at 13000 rpm (~16200 × g) for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.6. Add the solution obtained from steps 3 or 4 to the adsorption column that has been loaded into the collection tube, let it stand at room temperature for 2 minutes, centrifuge at 13000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.Attention: The volume of the adsorption column is 750 µ l. If the sample volume is greater than 750 µ L can be added in batches.7. Add 450 to the adsorption column µ LBuffer PW (please check if anhydrous ethanol has been added before use), centrifuge at 13000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.Note: If purified DNA is used for salt sensitive experiments (such as flat end ligation or direct sequencing), it is recommended to add Buffer PW and let it stand for 2-5 minutes before centrifugation.8. Repeat step 7.9.13000 rpm for 1 minute and discard the waste liquid from the collection tube.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).10. Place the adsorption column into a new 1.5 ml centrifuge tube (provided by oneself), and add 50 drops to the middle position of the adsorption membrane in the air µ L Buffer EB, leave at room temperature for 2 minutes. Centrifuge at 13000 rpm for 1 minute and collect DNA solution- Store DNA at 20 ℃.Attention:1) To improve the recovery of DNA, the solution obtained by centrifugation can be re dropped onto the adsorption column, left at room temperature for 2 minutes, and centrifuged at 13000 rpm for 1 minute.2) The elution volume should not be less than 30 µ l. A small volume will affect the recovery efficiency.3) When recovering DNA fragments larger than 10 kb, Buffer EB should be preheated in a 50 ℃ water bath to increase recovery efficiency.Note: This reagent kit is also suitable for the purification and recovery of PCR products. Add an equal volume of Buffer PG to the PCR reaction solution and mix thoroughly (for small fragments with a recovery of less than 150bp, the solution volume can be increased to three times to improve the recovery rate). Follow step 5 above for further operations... Read More | RAFT Agent Kit for controlling polymerizations at the molecular level detailed list of products: Catalog Number Product Name Component Catalog Number Component Name Component CAS Specification&Purity R396714 RAFT Agent Kit for controlling polymerizations at the molecular level C139356-500mg 4-RAFT Agent Kit for controlling polymerizations at the molecular level detailed list of products: Catalog Number Product Name Component Catalog Number Component Name Component CAS Specification&Purity R396714 RAFT Agent Kit for controlling polymerizations at the molecular level C139356-500mg 4-Cyano-4-(dodecylsulfanylthiocarbonyl)sulfanylpentanoic acid 870196-80-8 See Component Catalog Number R396714 RAFT Agent Kit for controlling polymerizations at the molecular level C396701-500mg Cyanomethyl dodecyl trithiocarbonate 796045-97-1 See Component Catalog Number R396714 RAFT Agent Kit for controlling polymerizations at the molecular level C396703-500mg Cyanomethyl methyl(phenyl)carbamodithioate 76926-16-4 See Component Catalog Number R396714 RAFT Agent Kit for controlling polymerizations at the molecular level C168358-500mg 2-Cyano-2-propyl benzodithioate 201611-85-0 See Component Catalog Number R396714 RAFT Agent Kit for controlling polymerizations at the molecular level C396706-500mg 2-(2-Cyanoprop-2-yl)-S-dodecyltrithiocarbonate 870196-83-1 See Component Catalog Number R396714 RAFT Agent Kit for controlling polymerizations at the molecular level C132316-500mg 4-Cyano-4-(phenylcarbonothioylthio)pentanoic Acid 201611-92-9 See Component Catalog Number R396714 RAFT Agent Kit for controlling polymerizations at the molecular level S396708-500mg S,S-Dibenzyl trithiocarbonate 26504-29-0 See Component Catalog Number R396714 RAFT Agent Kit for controlling polymerizations at the molecular level D396711-500mg 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid 461642-78-4 See Component Catalog Number... Read More | Product contentS666146Component50 T200 TStorageS666146ABuffer GR25 mL120 mLRTS666146BBuffer GL25 mL120 mLRTS666146CBuffer GW1 (concentrate)13 mL52 mLRTS666146DBuffer GW2 (concentrate)15 mL75 mLRTS666146EBuffer GE15 mL60 mLRTS666146FProteinase K1.25 mL4×1.25 mLRTS666146GSpin Columns DS with Product contentS666146Component50 T200 TStorageS666146ABuffer GR25 mL120 mLRTS666146BBuffer GL25 mL120 mLRTS666146CBuffer GW1 (concentrate)13 mL52 mLRTS666146DBuffer GW2 (concentrate)15 mL75 mLRTS666146EBuffer GE15 mL60 mLRTS666146FProteinase K1.25 mL4×1.25 mLRTS666146GSpin Columns DS with Collection Tubes50 sets 200 setsRTS666146HCentrifuge Tubes (1.5 mL)50 EA200 EARTProductsThis kit provides a simple and rapid method for the isolation and purification of total DNA from buccal swab samples. The kit adopts a silica matrix membrane that can specifically bind DNA and a unique buffer system to adsorb DNA efficiently and specifically, and 0.5-3.5 µg of genomic DNA can be obtained from each swab, and the extracted DNA fragments are large, pure and of stable and reliable quality. It is suitable for enzyme digestion, PCR, library construction, Southern hybridization and other experiments.Self-contained reagent: anhydrous ethanol.Pre-experiment Preparation and Important Notes1. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the label of the reagent bottle before first use.2. If precipitation is found in Buffer GL before use, dissolve Buffer GL in a 56°C water bath.3. All centrifugation steps can be performed at room temperature.4. Sampling: Use a buccal swab to wipe the inside of the mouth 6 times, dry for 2 hours and store. To ensure that the sample is not contaminated by food or drink, do not eat or drink for 30 minutes before sampling.Procedure1. The swab of the buccal swab was cut from the rod with scissors and placed in a 2mL centrifuge tube (supplied) and 400µL Buffer GR was added.Note: For genomic DNA without RNA contamination, add 4 µL of RNase A solution at a concentration of 100 mg/ml and shake to mix.2. Add 20 µL of Proteinase K and 400 µL of Buffer GL, immediately vortex and shake for 15 seconds and mix thoroughly.Note: Mix well immediately after adding Buffer GL; do not add Proteinase K directly to Buffer GL for use.3.56°C for 10 minutes and centrifuge briefly so that the solution on the walls of the tube collects at the bottom.4. Add 400 µL of anhydrous ethanol, vortex and shake to mix thoroughly, and centrifuge briefly so that the solution on the wall of the tube collects at the bottom of the tube.Note: The addition of anhydrous ethanol may produce a white precipitate that will not affect subsequent experiments.5. Add the solution and precipitate obtained in the previous step to the Spin Columns DS in two batches of up to 700 µL at a time into the collection tube. centrifuge the column at 12,000 rpm (∼13,400 × g) for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube.6. Add 500 µL of Buffer GW1 to the adsorbent 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 adsorbent 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 the column at 12,000 rpm for 3 minutes, pour off the waste liquid in the collection tube, and put the column back into the collection tube.Note: Step 7 can be repeated if further DNA purity is required.8. Centrifuge at 12,000 rpm for 1 minute 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 to remove residual ethanol from the adsorption column, which can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).9. Place the adsorption column in a new 1.5 mL centrifuge tube, add 50 µL of Buffer GE or sterilized water to the middle of the adsorption column overhanging the column, let stand at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store at -20℃.Attention:(1) If the downstream experiment is sensitive to pH or EDTA, it can be eluted with sterilized water. The pH value of the eluent has a great influence on the elution efficiency. If the eluent is made of water, the pH value should be 7.0-8.5 (the pH value of water can be adjusted to this range by using NaOH), and the elution efficiency is not high when the pH value is lower than 7.0.2) For long-term storage, it is recommended to elute with Buffer GE and store at -20°C... Read More |