| Description | Inquire | Product content： O665490Component50 TStorageO665490ABlocking Buffer500 mL2-8℃. Do not freeze.O665490BAntibody Pretreat Solution (HRP/Rabbit)5×1 mL2-8℃. Do not freeze.O665490CDilution Buffer500 mL2-8℃. Do not freeze.O665490DWash Buffer (10×)500 mL2-8℃. Do not Product content： O665490Component50 TStorageO665490ABlocking Buffer500 mL2-8℃. Do not freeze.O665490BAntibody Pretreat Solution (HRP/Rabbit)5×1 mL2-8℃. Do not freeze.O665490CDilution Buffer500 mL2-8℃. Do not freeze.O665490DWash Buffer (10×)500 mL2-8℃. Do not freeze. Product Introduction：The one-step rapid WB assay kit (rabbit) is the latest Western Blot detection kit developed by Kangwei Century, which canObtain high-quality Western Blot results within about 1 hour, with simple operation, high detection sensitivity, low background, and noAdditional secondary antibodies need to be added, with strong system stability. The conventional Western Blot indirect detection process (blocking, primary antibody binding)Combining with secondary antibodies requires a long time, a complex experimental process, and requires multi-step optimization of conditions. The protein on the glue is transferred toAfter coating the carrier membrane, incubate it with the blocking solution in the reagent kit for 5 minutes, and then incubate the carrier with the primary antibody treated with antibody reaction solutionAfter washing the membrane three times (5 minutes each time), it can undergo luminescence or color detection. This reagent kit is designed for target protein oneThe use of an experimental system derived from rabbits.Notes:1. Customers need to prepare their own rabbit source primary antibody.2. Before using Blocking Buffer blocking solution, Antibody Pretreat Solution (HRP/Rabbit) antibody reaction solution (rabbit), and Wash Buffer (10 x) rinse solution, please mix thoroughly.3. If there is precipitation in the rinsing solution when stored at 2-8 ℃, please restore it to room temperature, dissolve the precipitation, and use it normally. The 1x rinsing solution can be stored at room temperature for one month.4. It is recommended to stain the membrane with reagents such as spring red after the transfer is completed, and cut off any excess parts on the membrane to increase the efficiency of the reagents.5. The optimal dilution amount for primary antibody and antibody reaction solution HRP (rabbit) needs to be determined through preliminary experiments.6. Antibody reaction solution HRP (rabbit), antibody dilution solution, and antibody dosage can be increased or decreased proportionally according to the size of the membrane.7. The antibody dilution solution containing the first antibody can be recycled and reused once. Antibodies with low specificity and affinity are not recommended for repeated use. If the recovered antibody is used within 1-2 days and stored at 2-8 ℃ for long-term storage, please freeze it at -20 ℃ to avoid repeated freeze-thaw cycles.8. If there is a high background, please adjust the amount of antibodies and increase the number of times the film is washed.9. All reagents in the reagent kit should be stored at 2-8 ℃ to avoid freezing and thawing.Operation steps:This product is suitable for the sealing and antibody incubation steps after membrane transfer, taking a 5 cm x 8 cm membrane as an example:1. Preparation of rinsing solution: Dilute 10 ml of Wash Buffer (10 x) with distilled water to 100 ml, which is 1 x Wash Buffer. Set aside. Use 8-10 ml for each film wash.2. Sealing: After the membrane transfer is completed, immerse the membrane in 10 ml Blocking Buffer and seal at room temperature for 5 minutes.3. Rinse: Pour off the sealing solution, add 8-10 ml of 1 x Wash Buffer, and rinse at a high speed on a shaker for 1 minute.4. Prepare antibody incubation solution while washing the membrane: Take Antibody Pretreat Solution (HRP/Rabbit) 100 µ Add rabbit derived primary antibody 3-10 into the centrifuge tube µ g. Suck and beat the gun head until thoroughly mixed, and incubate at room temperature for 5 minutes. Add to 10 ml Dilution Buffer and mix well. Note: 1) The dosage of primary antibody can also be adjusted according to the dilution of the antibody. Taking the final dilution of antibodies at 1:1000 as an example, take 100 µ Add HRP (rabbit) antibody reaction solution into the EP tube and add 10 µ Add the first antibody to 10 ml of antibody diluent, mix well, and incubate at room temperature for 5 minutes. 2) If the membrane area is small, the amount of antibodies, reaction solution, and diluent can be reduced proportionally.5. After completing step 3, pour out the rinsing solution and add the antibody incubation solution mixed with primary antibody, Antibody Pretreat Solution (HRP/Rabbit), and Dilution Buffer to the membrane (ensuring that the incubation solution completely submerges the membrane surface). Incubate at room temperature on a shaker at a speed of about 60 rpm for 40 minutes.6. Discard (recover) the antibody incubation solution and rinse 3-5 times with the prepared 1 x Wash Buffer, each time for 3 minutes.7. Conduct subsequent testing. It is recommended to use ECL or DAB methods for testing.Example 1: Antigen 293T cell lysateA: Ordinary WB control: beta actin rabbit antibody (CW0097) 3.3ug incubated at room temperature for 40 minutes, washed with membrane, secondary antibody sheep anti rabbit HRP (CW0103) diluted at 1:10000, room temperature for 40 minutes, ECL (CW0049) exposed Example 2 Antigen is 293T cell lysateC: Ordinary WB control: PAK1, Epitomics rabbit monoclonal antibody 1:1000, incubated at room temperature for 40 minutes, washed with membrane, secondary antibody sheep anti rabbit HRP (CW0103) diluted at 1:10000, room temperature for 40 minutes, ECL (CW0049) exposedD: One step WB: Epitomics rabbit monoclonal antibody was incubated at 1:1000 room temperature for 40 minutes, and ECL (CW0049) was exposed... 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 | Inquire | Product contentY666144Component50 TStorageY666144ABuffer P115 mLRTY666144BBuffer P215 mLRTY666144CBuffer N320 mLRTY666144DBuffer PS15 mLRTY666144EBuffer PB10 mLRTY666144FBuffer PW (concentrate)10 mLRTY666144GBuffer EB10 mLRTY666144HGlass Beads2 gRTY666144IRNase A (10mg/mL)150 µLRTY666144JSpin Product contentY666144Component50 TStorageY666144ABuffer P115 mLRTY666144BBuffer P215 mLRTY666144CBuffer N320 mLRTY666144DBuffer PS15 mLRTY666144EBuffer PB10 mLRTY666144FBuffer PW (concentrate)10 mLRTY666144GBuffer EB10 mLRTY666144HGlass Beads2 gRTY666144IRNase A (10mg/mL)150 µLRTY666144JSpin Columns DM with Collection Tubes50 setsRTProductsThis kit is improved on the basis of common alkaline lysis method, the glass beads can effectively break the yeast cell wall, the new silica matrix membrane and buffer system can efficiently and specifically bind the plasmid DNA, and at the same time can maximize the removal of proteins and other impurities, the whole process is convenient and fast, no need to use toxic and harmful reagents, and can be processed at the same time for multiple samples. In addition to yeast cells, it can also be used in E. coli. Plasmid DNA extracted with this kit can be used in various molecular biology experiments, such as ligation, transformation, sequencing and library screening.Self-contained reagents: β-mercaptoethanol, anhydrous ethanol.Pre-experiment Preparation and Important Notes1. All components can be stably stored in dry, room temperature (15-30℃) environment for 1 year, the adsorption column can be stored at 2-8℃ for a longer period of time, and Buffer P1 with RNase A can be stably stored at 2-8℃ for 6 months.2. Before the first use, add all the RNase A solution to Buffer P1, mix well, and store at 2-8℃.3. Anhydrous ethanol should be added to Buffer PW before first use according to the instructions on the reagent bottle label.4. Before use, please check whether Buffer P2 and Buffer N3 are crystallized or precipitated. If there is any crystallization or precipitation phenomenon, it can be clarified by taking a water bath at 37℃ for a few minutes to restore the clarity.5. Be careful not to touch Buffer P2 and Buffer N3 directly, and tighten the lid immediately after use.6. The amount of plasmid extracted is related to the yeast strain, plasmid copy number, culture conditions, etc. Usually, yeast plasmid copy number is very low, which is difficult to be detected by electrophoresis or spectrophotometer method.Procedure1. Take 1-5 ml of yeast culture (maximum 5×107 yeast cells, generally for Saccharomyces cerevisiae OD = 1.0, equivalent to 1-2×107 cells/ml) and add it to a centrifuge tube (self-provided), centrifuge for 30 seconds at 12,000 rpm (~13,400×g), collect the bacterial precipitate, and aspirate as much as possible to discard the supernatant.2. Add 250µl Buffer P1 to the bacterium (please check if RNase A has been added first) and resuspend the precipitate.3. Add 40mg of Glass Beads to the above mixture and vortex and shake for 10 minutes.4. Add 250 µl of Buffer P2 to the centrifuge tube, mix gently by turning up and down 6-8 times, and let stand at room temperature for 5-10 minutes, at which time the bacterial solution should become clear and viscous.Note: Mix gently, do not shake violently, so as not to interrupt the genomic DNA, resulting in genomic DNA fragments mixed in the extracted plasmid. If the solution does not become clear, it suggests that the amount of bacteria may be too large and the lysis is not complete, and the amount of bacteria should be reduced.5. Add 350 µl of Buffer N3 to the centrifuge tube and immediately mix gently up and down 6-8 times, at which point a white flocculent precipitate appears, and centrifuge at 12,000 rpm for 20 minutes.Note: Buffer N3 should be mixed immediately after addition to avoid localized precipitation.6. Column Equilibration: Add 200 µl of Buffer PS to the Spin Columns DM in the collection tube, centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and place the column back into the collection tube.7. Add the supernatant from step 5 to the adsorbent column that has been loaded into the collection tube, taking care not to aspirate the precipitate.Note: The maximum volume of the adsorption column is 750 µl, and the solution is passed through the column in 2 times.8. Centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube and place the adsorption column back into the collection tube.9. Add 150 µl Buffer PB to the adsorbent column, 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.10. Add 750 µl Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first), centrifuge at 12,000 rpm for 1 minute, and pour off the waste liquid in the collection tube.11. Place the column back into the recovery collection tube and centrifuge at 12,000 rpm for 2 minutes, pouring off the waste liquid. Leave the 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; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).12. Place the adsorbent column in a new centrifuge tube, add 50-100 µl of Buffer EB to the center of the adsorbent membrane dropwise, let it stand at room temperature for a few minutes, centrifuge at 13,000 rpm for 1 minute, and collect the plasmid solution into the centrifuge tube. Store the plasmid at -20°C.Attention:1) To increase the recovery efficiency of the plasmid, the resulting solution can be reintroduced into the adsorbent column, left at room temperature for a few minutes, centrifuged at 13,000 rpm for 1 minute, and the plasmid solution collected into a centrifuge tube.2) When the plasmid copy number is low or >10 kb, Buffer EB is preheated at 65-70°C in a water bath, which can increase the extraction efficiency.3) Usually yeast plasmids have very low copy number and are difficult to detect by electrophoresis or spectrophotometry. If the extracted plasmid is to be used in the next step of the experiment, it is usually recommended to use 1-5µl of the plasmid as PCR template, and 5-10µl of the plasmid for transformation of E. coli.4) Commercial high transformation efficiency receptor cells should be used for transformation of E. coli... Read More |