| Description | Inquire | Product content： Component O66550510 preps O665505 50 preps Blocking Buffer 100 ml 500 ml Antibody Pretreat Solution（ HRP/Mouse ） 1 ml 5 × 1 ml Dilution Buffer 100 ml 500 ml Wash Buffer（ 10× ) 100 ml 500 mlProduct Introduction:The one-step rapid WB assay kit (Product content： Component O66550510 preps O665505 50 preps Blocking Buffer 100 ml 500 ml Antibody Pretreat Solution（ HRP/Mouse ） 1 ml 5 × 1 ml Dilution Buffer 100 ml 500 ml Wash Buffer（ 10× ) 100 ml 500 mlProduct Introduction:The one-step rapid WB assay kit (mouse) is the latest Western Blot detection kit developed by Kangwei Century, which can obtain high-quality Western Blot results in about 1 hour. It is easy to operate, has high detection sensitivity, low background, does not require the addition of secondary antibodies, and has strong system stability. The conventional Western Blot indirect detection process (blocking, primary antibody binding, and secondary antibody binding) requires a long time, a complex experimental process, and requires multi-step condition optimization. After transferring the protein on the gel to the carrier membrane, incubate it with the blocking solution in the reagent kit for 5 minutes, and then incubate the carrier membrane with the primary antibody treated with antibody reaction solution. After washing three times (5 minutes each time), luminescence or colorimetric detection can be performed. This reagent kit is designed for use in experimental systems where the target protein primary antibody is derived from mice.Notes:1. The customer prepares their own mouse source primary antibody.2. Before using Blocking Buffer blocking solution, Antibody Pretreat Solution (HRP/Mouse) antibody reaction solution (mouse), 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 (mouse) needs to be determined through preliminary experiments.6. The antibody reaction solution HRP (mouse), 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. It is recommended not to reuse antibodies with poor specificity and affinity. 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/Mouse) 100 µ Add mouse 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 (mouse) 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/Mouse), and Dilution Buffer to the membrane (ensuring that the incubation solution completely submerges the surface of the membrane). Incubate at room temperature on a shaker at around 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.Application examples:Example 1 Antigen is 293T cell lysateA: Normal WB control: beta actin mouse monoclonal antibody (CW0096) 5 µ Incubate at room temperature for 40 minutes, wash the film and dilute the secondary antibody sheep anti mouse HRP (CW0102) 1:10000. Incubate at room temperature for 40 minutes and expose ECL (CW0049).B: One step method WB: beta actin mouse monoclonal antibody (CW0096) 5 µ Incubate at room temperature for 40 minutes and expose ECL (CW0049).Example 2 Antigen is E. coli multi label protein lysateC: Normal WB control: GST mouse monoclonal antibody (CW0084) 2.5 µ Incubate at room temperature for 40 minutes, wash the film and dilute the secondary antibody sheep anti mouse HRP (CW0102) 1:10000. Incubate at room temperature for 40 minutes and expose ECL (CW0049).D: One step method WB: GST mouse monoclonal antibody (CW0084) was incubated at room temperature with 2.5ug for 40 minutes, and ECL (CW0049) was exposed... Read More | Product contentP666142Component200 TStorageP666142ABuffer P160 mLRTP666142BBuffer P260 mLRTP666142CBuffer N380 mLRTP666142DBuffer PB35 mLRTP666142EBuffer PW (concentrate)25 mLRTP666142FBuffer EB30 mLRTP666142GRNase A (10 mg/mL)600 µLRTP666142HSpin Columns DM with Collection Tubes200 EART Product contentP666142Component200 TStorageP666142ABuffer P160 mLRTP666142BBuffer P260 mLRTP666142CBuffer N380 mLRTP666142DBuffer PB35 mLRTP666142EBuffer PW (concentrate)25 mLRTP666142FBuffer EB30 mLRTP666142GRNase A (10 mg/mL)600 µLRTP666142HSpin Columns DM with Collection Tubes200 EART Product IntroductionThis kit is suitable for extracting 1-5 ml of bacterial solution. Based on the lysis of cells by alkaline lysis method, it adopts a unique silica matrix membrane adsorption technology and reagent formulation, and efficiently and exclusively binds plasmid DNA in solution by centrifugal adsorption columns in a high-salt state, and each adsorption column can adsorb a maximum of 30 µg of plasmid DNA, and removes proteins, genomes, RNAs, and other impurities to the greatest extent possible. The plasmid DNA obtained can be directly used for cell transfection, PCR, digestion, sequencing, ligation and other biological experiments.Self-contained reagent: 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 into Buffer P1, mix well, and store it at 2-8°C. Before use, leave it at room temperature for a period of time, and then use it after recovering to room temperature.3. Anhydrous ethanol should be added to Buffer PW according to the instructions on the label of the reagent bottle before first use.4. If precipitation is found in Buffer P2, Buffer N3, or Buffer PB before use, the clarification can be restored by water bath at 37℃ for a few minutes (please do not shake Buffer P2 violently).5. Be careful not to touch Buffer P2, Buffer N3 and Buffer PB directly, and tighten the lid immediately after use.6. The amount and purity of extracted plasmid is related to the concentration of bacterial culture, strain type, plasmid size, plasmid copy number and other factors.Procedure1. Take 1-5 ml of the overnight culture and add it to a centrifuge tube (self-prepared), centrifuge for 30 seconds at 13,000 rpm (~16,200×g) to collect the bacterial precipitate, and discard the supernatant as much as possible.2. Add 250 µl of Buffer P1 to the centrifuge tube with the bacterial precipitate (please check that RNase A has been added first), mix well using a pipette or vortex shaker, and suspend the bacterial precipitate.Note: If the bacterial mass is not thoroughly mixed, it will affect the lysis effect, resulting in low extraction and purity.3. Add 250µl of Buffer P2 to the centrifuge tube and mix gently up and down 4-6 times, mixing well to lyse the organisms, at which point the solution should become clear and viscous.Note: Mix gently, do not shake vigorously to avoid interrupting the genomic DNA and causing the extracted plasmid to be mixed with genomic DNA fragments. This step should take no more than 5 minutes to avoid damage to the plasmid.4. Add 350 µl of Buffer N3 to the centrifuge tube and immediately mix gently up and down for 8-10 times, mixing well so that a white flocculent precipitate should appear. centrifuge at 13,000 rpm for 5 minutes.Note: Buffer N3 should be mixed immediately after addition to avoid localized precipitation.5. Transfer the supernatant obtained in step 4 to the Spin Columns DM that have been loaded into the collection tube, centrifuge at 13,000 rpm for 30 seconds, pour off the waste liquid from the collection tube, and place the column back into the collection tube.6. Add 150 µl Buffer PB to the adsorption column and centrifuge at 13,000 rpm for 30 seconds.7. Add 400 µl Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first), centrifuge at 13,000 rpm for 1 minute, and pour off the waste liquid in the collection tube.8. Place the adsorbent column in a new centrifuge tube (supplied), add 50-100 µl Buffer EB to the middle of the adsorbent membrane, leave it at room temperature for 2 minutes, centrifuge at 13,000 rpm for 1 minute, and collect the plasmid solution into the centrifuge tube. -The plasmid solution was collected into the centrifuge tube.Note: 1) To increase the recovery efficiency of the plasmid, the resulting solution can be reintroduced into the adsorbent column, left at room temperature for 2 minutes, centrifuged at 13,000 rpm for 1 minute, and the plasmid solution collected into a centrifuge tube.2) For low plasmid copy number or >10 kb, Buffer EB is preheated at 65-70°C in a water bath to increase extraction efficiency... Read More | Products contentProducts IntroductionThis product uses the principle that the difference between the concentration of salt ions inside and outside the cell can cause the cell membrane to burst to lyses the cell and releases the genomic DNA, without the need of extracting and purifying the genomic Products contentProducts IntroductionThis product uses the principle that the difference between the concentration of salt ions inside and outside the cell can cause the cell membrane to burst to lyses the cell and releases the genomic DNA, without the need of extracting and purifying the genomic DNA.This product is suitable for a variety of sources of samples, and can be used as a template for PCR and qPCR experiments after sample processing, and can achieve the effect of the purified DNA used as a template for PCR and qPCR experiments. Usage1. Depending on the type of sample, prepare the appropriate sample size according to the table below.2. Add the sample to a 1.5-mi centrifuge tube and add the recommended volume of Solution A as shown in the table below. Vortex for 20 s and allow to stand at room temperature for 3-5 min or incubate in a metal bath at 95°C for 3-5 min as recommended in the table below.3. After the sample has been sufficiently lysed (samples incubated in a metal bath at 95°C should be brought to room temperature), add the recommended volume of Solution B as shown in the table below and vortex for 30s.4. Store processed samples at 4°C if the next test is to be performed within 2 hours, or at -20°C if the next test cannot be performed immediately.take note of1) Depending on the requirements of the experimental conditions, the amount of samples can be expanded or reduced, and the amount of Solution A and Solution B can be increased in equal proportions.2) For blood and cell samples, the temperature of room temperature lysis is required to be around 25C. If the ambient temperature does not reach 25°, the lysis time can be extended appropriately, or the vortex shaking time can be extended to ensure that the samples are fully lysed. If there is no relevant professional instrument, the centrifuge tube can be shaken vigorously to ensure adequate lysis.3) After the tissue sample is made into tissue homogenate by adding 10 times the volume of saline, it can be processed in the same way as blood samples.4) Strictly prohibit the use of expired products, please do not mix different reagents.5) laboratory supplies should be regularly cleaned and 10% of the 84 disinfectant solution or ultraviolet lamp for anti-pollution treatment, special areas dedicated to prohibit cross use, so as to avoid contamination, the end of the test, the bench should be cleaned immediately... Read More | Product DescriptionAcetyl esterase (sialate-O-acetylesterase) is a recombinant protein from Tannerella forsythia, ATCC 43037 strain, expressed in Escherichia coli. The enzyme removes acetyl groups attached via an O- group, mainly 9-, 8- and 7-. It can be used for monitoring of diacetylation of Product DescriptionAcetyl esterase (sialate-O-acetylesterase) is a recombinant protein from Tannerella forsythia, ATCC 43037 strain, expressed in Escherichia coli. The enzyme removes acetyl groups attached via an O- group, mainly 9-, 8- and 7-. It can be used for monitoring of diacetylation of sialic acids on products such as erythropoietin (EPO).The Zyme Acetyl Esterase Kit removes 9-, 8- and 7-O-acetyl groups from released sialic acids, released glycans or glycoproteins. It is commonly used for the characterization of highly-sialylated biotherapeutics such as EPO, FSH and blood clotting factors.Molecular Weight76.3 kDContentsAcetyl esterase – PBS pH7.5 buffer containing 10 mM Tris-HClReaction Buffer – 500 mM sodium acetate pH5.5Number of SamplesSufficient for up to 50 samples.Amount of SampleUp to 10 µg glycoprotein, up to 2.5 µg released glycans and up to 1 µg free sialic acid per digestion.Suitable SamplesAcetyl esterase (sialate-O-acetylesterase) can act upon complex glycoprotein samples, such as erythropoietin (EPO), bovine submaxillary mucin and oral epithelial cell-bound glycans, and on N- and O-glycans released from a glycoprotein. Either fluorescently labelled or unlabelled glycans are suitable. It can also be used on released sialic acids.Unit DefinitionOne unit (U) of acetyl esterase is defined as the amount of enzyme required to produce 300 µmole of 4-nitrophenol and acetate in 1 minute at 30°C in a buffer containing 50 mM Tris-HCl, 140 mM NaCl, pH 8.5, from 4-nitrophenyl acetate, a chromogenic esterase substrateStorageProtect from sources of heat and light. When stored correctly, the enzyme should be stable for 24 months from date of purchase. Exposure to ambient temperatures (20 – 26°C) over 3 days does not result in a reduction of enzymatic activity.ShippingThe product should be shipped at 4°C.HandlingEnsure that any glass, plastic ware or solvents used with this item are free of environmental carbohydrates. Use powder-free gloves for all sample handling procedures and avoid contamination with environmental carbohydrate.SafetyPlease read the Safety Data Sheets (SDSs) for all chemicals used. All processes involving labelling reagents should be performed using appropriate personal safety protection – safety glasses, chemically resistant gloves (e.g. nitrile), lab coat, and when appropriate, in a laboratory fume cupboard.For research use only. Not for human or drug use ApplicationAcetyl esterase (sialate-O-acetylesterase) can be used to remove 9-, 8- and 7-O-acetyl groups from released sialic acids, released glycans or glycoproteins... Read More |