| Description | Bacterial protein extraction reagents use mild non-ionic detergents and are suitable for extracting recombinant proteins expressed in Escherichia coli and insect cells. During the extraction process, there is no need for ultrasonic fragmentation, effectively avoiding contamination of exogenous Bacterial protein extraction reagents use mild non-ionic detergents and are suitable for extracting recombinant proteins expressed in Escherichia coli and insect cells. During the extraction process, there is no need for ultrasonic fragmentation, effectively avoiding contamination of exogenous proteins. This product can be applied to extract soluble proteins from bacterial lysates. The bacterial protein extraction kit adds a mixture of lysozyme, DNase I, and protease inhibitors to the extraction reagent, which can improve the efficiency of protein extraction and reduce the viscosity caused by DNA, effectively avoiding protein degradation. The extracted protein maintains biological activity and can be subjected to downstream operations such as IP, Western blot, and protein purification. Component B665764 100 preps Bacterial Protein Extraction Reagent 100 ml Protease Inhibitor Cocktail (100x) 1 ml Lysozyme （50 mg/ml） 200µl DNaseⅠ（1,000 U/ml） 100µl Notes:1. This product is suitable for extracting proteins from fresh or frozen bacterial and insect cells.2. This product uses Tris buffer system. Please use the same buffer system for protein purification after extraction.3. The protein lysis solution obtained from this product can be used for protein quantification using BCA or Bradford method.4. For special strains, if the extraction effect is not ideal, the sample can be frozen before protein extraction.5. Depending on the specific situation, protease inhibitors, salts, chelating agents, reducing agents, etc. can be added to this product.Operation steps: ● Insect cell protein extraction1. Collect cells by low-speed centrifugation. Add 10 to every 1 ml of Bacterial Protein Extraction Agent µ The Protein Inhibitor Cocktail is 1 x working fluid.2. Weigh the wet weight of the cells and add 1 x working solution at a rate of 10 ml/g.3. After resuspension, incubate on ice for 20 minutes (the ice storage time should be adjusted according to different cell types).Centrifuge at 4.15000 × g for 15 minutes to isolate soluble proteins. ● Extraction of soluble bacterial proteins 1. Centrifuge for 10 minutes at a rate of 5000 × g and collect the bacterial cells.2. Optional steps: Add 1 ml of Bacterial Protein Extraction Reagent every 1 ml µ DNase I (1000 U/ml), 2 µ Lysozyme (50 mg/ml) and 10 µ Protein Inhibitor Cocktail, vortex oscillation and mixing. 3. Add 20 ml of Bacterial Protein Extraction Reagent to each gram of bacterial precipitate, and add the extraction solution to the bacterial precipitate. Vortex thoroughly or use a pipette to blow up and down until the bacterial precipitate is completely resuspended.4. After resuspension, incubate at room temperature for 10-15 minutes (the storage time should be adjusted according to different cell types). 5. Centrifuge at 15000 × g for 5 minutes.6. Transfer the supernatant to a new centrifuge tube (the supernatant is soluble protein) for protein quantification and downstream experiments.Note: If the target protein exists in the form of inclusion bodies, inclusion body protein solution can be used for dissolution or expression conditions can be optimized to increase the expression of soluble proteins.Frequently asked questions: Problem Possible reasons Resolvent The target protein is insoluble The target protein is expressed as an inclusion body Optimize expression conditions or add Lysozyme and DNase I to protein extraction reagents using inclusion body protein solution After adding Lysozyme, the target protein has not been extracted yet Temperature too low Restore the reagent to room temperature After adding Lysozyme, the target protein has not been extracted yet Lysozyme Decreased or inactivated activity Add more Lysozymes or replace with new enzymes Extract has high viscosity DNase I Decreased or inactivated activity Add more DNase I or replace with a new DNase I to increase the final concentration of magnesium ions to 2 mM After protein extraction, most of the proteins still exist in the precipitate Excessive protein content Add Lysozyme and DNase I The protein extraction reagent has sediment precipitation Temperature too low Restore the protein extraction reagent to room temperature... Read More | FFPE DNA/RNA KitFixed Tissue DNA/RNA Extraction Kit Catalog number: F666120 (50 preps)Storage conditions: DNase I and 10×Reaction Buffer -20℃, Spin Columns DF and Spin Columns RS can be stored at room temperature for 2 months, 2-8℃ for 1 year, the rest of the components are stored FFPE DNA/RNA KitFixed Tissue DNA/RNA Extraction Kit Catalog number: F666120 (50 preps)Storage conditions: DNase I and 10×Reaction Buffer -20℃, Spin Columns DF and Spin Columns RS can be stored at room temperature for 2 months, 2-8℃ for 1 year, the rest of the components are stored at room temperature (15-30℃).Products Content:Products IntroductionThis kit is suitable for the effective purification of genomic DNA and total RNA from paraffin-embedded tissues, using specially optimized deparaffinizing agents and lysates to release DNA and RNA from tissue section samples, without the use of the organic reagent xylene, and without the need for overnight operation; the digested samples are incubated at higher temperatures to remove inhibitors caused by cross-linking, which can effectively improve nucleic acid yields and purity; and an optimized buffer system allows nucleic acids in the lysate to bind specifically to the adsorbent membrane, and inhibitors are effectively removed by a two-step rinsing procedure. The optimized buffer system enables the nucleic acids in the lysate to bind specifically to the adsorbent membrane, and the inhibitors are effectively removed by a two-step rinsing step, and finally eluted with low-salt buffer or water to obtain high purity DNA and RNA, and at the same time, equipped with a high-efficiency microsorbent column, the volume of the elution can be as low as 20 µl. The purified DNA and RNA can be directly used for PCR, Real-time PCR, SNP genotyping, STR genotyping, and so on. The purified DNA and RNA can be directly used for PCR, Real-time PCR, SNP genotyping, STR genotyping, second-generation sequencing, pharmacogenomics research and blot analysis.Self-contained reagent: anhydrous ethanolPre-experiment Preparation and Important Notes 1. After obtaining the sample, fix the sample as soon as possible, the fixation time of 14-24 hours is appropriate, too long a period of time will easily lead toDNA and RNA breaks, affecting downstream experiments. If the formaldehyde fixation time is too long or the sample is stored for too long(>1 year) is prone to compromise DNA integrity and failure to amplify long fragments.2. Ensure that samples are thoroughly dehydrated prior to embedding; residual formalin will inhibit Proteinase K action.3. Add 1.25 ml of Proteinase K Storage Buffer to Proteinase K to dissolve it, and store at -20℃. Do not leave the prepared Proteinase K at room temperature for a long period of time to avoid affecting its activity.4. Anhydrous ethanol should be added to Buffer RW2, Buffer GW1 and Buffer GW2 according to the label instructions on the vials before first use.5. Check Buffer GTL, Buffer GL and Buffer DS for crystallization or precipitation prior to use; if crystallization or precipitation occurs, redissolve Buffer GTL, Buffer GL and Buffer DS in a 37°C water bath.6. Preheat the water bath or thermostatic mixer to 56°C before starting the experiment.7. Use an ambient temperature centrifuge or set the centrifuge temperature to 25°C. Temperatures below 15°C may result in clogging of the adsorption column.8. To prevent RNase contamination, the following should be observed:1) Use RNase-free plastics and tips to avoid cross-contamination.(2) Glassware should be dry baked at 180°C for 4 hours before use, plasticware can be soaked in 0.5 M 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.procedureParaffin-embedded samples1. Trim off excess paraffin from the tissue block to expose the tissue and cut into 5-10 µm slices.2. Place approximately 1 x 1 cm2 slices (1-5 slices in total) in a centrifuge tube (supplied), add 500 µl of Buffer DS and vortex for 10 s. Briefly centrifuge the sample to the bottom of the tube. Centrifuge briefly to collect the sample at the bottom of the tube, incubate at 56°C for 3 minutes, remove from the water bath and allow to cool to room temperature before proceeding.Note: If the surface of the sample is exposed to air, discard the initial 2-3 slices without using them.3. Centrifuge at 12,000 rpm for 2 minutes and carefully discard the supernatant thoroughly without aspirating the precipitate. The residual dewaxing solution can be carefully removed with a small tip (10 µl).4. Add 180 µl of Buffer GTL and 20 µl of Proteinase K to the above tube and mix well with vortexing.5. Incubate at 56°C for 15 minutes, then place on ice for 3 minutes. Centrifuge at 12,000 rpm for 15 minutes at room temperature.6. Transfer the supernatant to a new 1.5 ml centrifuge tube for RNA extraction, taking care not to aspirate undigested tissue. Use the precipitate for DNA extraction. RNA extraction7. Take the supernatant obtained in step 6 and incubate at 80°C for 15 minutes.8. Add 320 µl of Buffer GL, mix by vortexing and shaking, then add 720 µl of anhydrous ethanol and mix immediately by vortexing and shaking.9. Add all of the resulting solution to the Spin Columns RS in the collection tube; if the solution cannot be added all at once, it may be transferred in several passes. centrifuge the column at 12,000 rpm for 1 minute, pour off the waste solution from the collection tube, and place the column back into the collection tube. Note: If the columns are clogged, the sample size may be too large and consideration should be given to reducing the number of starting sections to 1-2.Optional step: If genomic DNA is to be removed, the following steps can be followeda. Add 350 µl of Buffer RW1 to the column, centrifuge at 12,000 rpm for 1 minute, discard the waste solution, and place the column back into the collection tube.b. Preparation of DNase I mixture: Take 52 µl of RNase-Free Water and add 8 µl of 10×Reaction to it.Buffer and 20 µl DNase I (1 U/µl), mix well, and prepare a final volume of 80 µl of reaction solution.c. Add 80 µl of DNase I Mix directly to the adsorption column and incubate at 20-30°C for 15 minutes.d. Add 350 µl of Buffer RW1 to the column, centrifuge at 12,000 rpm for 1 minute, discard the waste solution, and return the column to the collection tube.Add 500 µl of Buffer RW2 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.11. Repeat step 10. centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Place the column at room temperature for 5 minutes.minutes to dry thoroughly.12. Place the column in a new RNase-free centrifuge tube and add 20-50 µl to the center of the column.RNase-Free Water, left at room temperature for 5 minutes, centrifuged at 12,000 rpm for 1 minute, and collected RNA solution, the-80°C for storage.DNA extraction7. Take the precipitate obtained in step 6 and add 180 µl Buffer GTL and 20 µl Proteinase K to the precipitate. VortexResuspend the precipitate for 15 seconds.8. Incubate at 56°C for 1 hour until the sample is completely dissolved. 90°C for 1 hour.Add 200 µl Buffer GL, vortex and shake to mix and then add 200 µl anhydrous ethanol, vortex and shake to mix thoroughly. Centrifuge briefly so that the solution on the wall of the tube collects at the bottom of the tube. Add all of the solution from step 9 to the Spin Columns DF in the collection tube, or transfer the solution in several passes. centrifuge at 12,000 rpm for 1 minute, pour off the waste solution from the collection tube, and return the column to the 10. collection tube.Note: If the adsorption column is clogged, the sample size may be too large and consideration should be given to reducing the number of starting sections to 1-2.11. Add 500 µl of Buffer GW1 to the adsorbent column and centrifuge at 12,000 rpm for 1 minute. Pour off the waste liquid from the collection tube and put the column back into the collection tube.12. Add 500 µl of Buffer GW2 to the adsorbent column and 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.Note: Step 12 may be repeated if further purity is required.13. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Leave the adsorption column at room temperature for 5 minutes to dry thoroughly. Note: The purpose of this step is to remove residual ethanol from the adsorbent column; ethanol residue will affect the subsequent enzymatic reaction. 14. Place the column in a new 1.5 ml centrifuge tube, add 20-50 µl Buffer EB to the center of the column, leave at room temperature for 5 minutes, centrifuge at 12,000 rpm for 1 minute, collect the DNA solution, and store at -20℃... Read More | This reagent kit is for research purposes only. Purpose of use: This reagent kit is used to determine the content of lactose in serum, plasma, and related liquid samples.Experimental principle:This kit applies enzyme-linked immunosorbent assay to determine the level of lactose in the sample. This reagent kit is for research purposes only. Purpose of use: This reagent kit is used to determine the content of lactose in serum, plasma, and related liquid samples.Experimental principle:This kit applies enzyme-linked immunosorbent assay to determine the level of lactose in the sample. Purified lactose antibodies were coated on a microplate to produce solid-phase antibodies. Lactose was added to the microplate of the coated monoclonal antibody, along with HRP labeled lactose antigens, to compete for binding. After thorough washing, the substrate TMB was added for colorimetry. The depth of sample color is negatively correlated with the content of lactose in the sample. Measure the absorbance (OD value) at a wavelength of 450nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the content of lactose in the sample through a standard curve.Kit composition:130times concentrated washing solution20ml×1 bottle8.1Standard S1（80µg/L）0.5ml×1bottle2Enzyme-linked immunosorbent assay6ml×1 bottle8.2Standard S2（40µg/L）0.5ml×1bottle3Enzyme labeling coated plate96 holes x 1 pieces8.3Standard S3（20µg/L）0.5ml×1bottle4Color reagent A solution6ml×1 bottle8.4Standard S4（10µg/L）0.5ml×1bottle5Color developer B solution6ml×1 bottle8.5Standard S5（5µg/L）0.5ml×1bottle6Stop solution6ml×1 bottle9Instructions1 copy7Sample Diluent6ml×1 bottle10Microplate Sealers2 sheetsSpecimen requirements:1. Specimen processing:(1) After collecting the water sample, it is repeatedly freeze-thawed three times at -20 ℃, and then filtered through glass fiber for future reference(2) The tissue samples should be extracted using butanol: methanol: water (5:25:70 V: V: V), or extracted according to relevant literature. The experiment should be conducted as soon as possible after extraction. If the experiment cannot be conducted immediately, the specimen can be stored at -20 ℃ for future reference2. Samples containing NaN3 cannot be detected as NaN3 inhibits the activity of horseradish peroxidase (HRP).Operation steps:1. Sample addition: Set up standard wells, blank wells (blank control wells do not include samples and enzyme-linked immunosorbent assay reagents, the other steps are the same), and sample wells to be tested. Add 50 microliters to the standard well on the enzyme-linked immunosorbent assay (ELISA) plate, and first add 40 diluents to the sample well to be tested µ l. Then add 10 more samples to be tested µ L (The final dilution of the sample is 5 times). Add the sample to the bottom of the enzyme-linked immunosorbent assay (ELISA) plate well, avoiding touching the well wall as much as possible. Gently shake and mix well.2. Enzyme addition: Add 50 enzyme labeled reagents to each well µ l. Excluding blank holes.3. Warm incubation: Seal the plate with a sealing film and incubate at 37 ℃ for 60 minutes.4. Solution preparation: Dilute 30 times the concentrated washing solution with distilled water and set aside for later use5. Washing: Carefully remove the sealing film, discard the liquid, shake dry, fill each well with washing solution, let it stand for 30 seconds, then discard. Repeat this process 5 times and pat dry.6. Color development: Add color development agent A50 to each well first µ l. Add color developer B50 again µ l. Gently shake and mix well, and develop color at 37 ℃ in the dark for 15 minutes7. Termination: Add 50% termination fluid to each hole µ l. Terminate the reaction (at this point, the blue color immediately turns yellow).8. Measurement: Zero the blank hole and sequentially measure the absorbance (OD value) of each hole at a wavelength of 450nm. The measurement should be conducted within 15 minutes after adding the termination solution.Calculation:Draw a standard curve on a coordinate paper with the concentration of the standard substance as the x-axis and the OD value as the y-axis. Based on the OD value of the sample, determine the corresponding concentration from the standard curve; Multiply it by the dilution factor; Alternatively, a linear regression equation can be used to calculate the standard curve using the concentration and OD value of the standard substance. The OD value of the sample can be substituted into the equation to calculate the sample concentration, which is then multiplied by the dilution factor to obtain the actual concentration of the sample.Notes:1. The kit should be balanced at room temperature for 15-30 minutes before use when taken out from the cold storage environment. If the enzyme coated plate is not used up after opening, the Flat noodles should be stored in a sealed bag.2. Concentrated detergent may precipitate crystals. When diluted, it can be heated in a water bath to aid in dissolution. Washing does not affect the results.3. A sampler should be used for each step of sample addition, and its accuracy should be regularly calibrated to avoid experimental errors. It is best to control the sample addition time within 5 minutes. If there are a large number of specimens, it is recommended to use a firing gun for sample addition.4. Please make a standard curve at the same time as each measurement, preferably with a re hole. If the content of the substance to be tested in the sample is too high (the OD value of the sample is greater than the OD value of the first well of the standard well), please dilute the sample diluent by a certain multiple (n times) before measurement. When calculating, please multiply the total dilution multiple (x n x 5).5. The sealing film is only for one-time use to avoid cross contamination.6. Please store the substrate in dark.7. Strictly follow the instructions and determine the test results based on the reading of the enzyme-linked immunosorbent assay (ELISA) reader8. All samples, washing liquids, and various waste should be treated as infectious substances.9. The components of this reagent with different batch numbers shall not be mixed.Detection range:two µ G/L-90 µ G/L... Read More | Inquire | This kit combines efficient guanidine isothiocyanate lysis technology with silicon matrix membrane purification technology to efficiently extract total RNA from animal cells and tissues. The starting sample usually has a maximum of 30 mg of tissue or 1 x 107 cells. This reagent kit can also recover This kit combines efficient guanidine isothiocyanate lysis technology with silicon matrix membrane purification technology to efficiently extract total RNA from animal cells and tissues. The starting sample usually has a maximum of 30 mg of tissue or 1 x 107 cells. This reagent kit can also recover partially purified RNA, RNA obtained from in vitro transcription and enzymatic reactions. This reagent kit can extract and purify high-quality RNA with a molecular weight greater than 200 bases, with almost no DNA residue. If RNA experiments are to be conducted that are highly sensitive to trace amounts of DNA, residual DNA can be digested and removed on a column using DNase I without RNase. The extracted RNA can be used for downstream experiments such as RT-PCR, Northern Blot, Dot Blot, etc. R666020Component50 TStorageR666020ABuffer RL35 mLRTR666020BBuffer RW140 mLRTR666020CBuffer RW2 (concentrate)11 mLRTR666020DRNase-Free Water10 mLRTR666020ESpin Columns RM with Collection Tubes50 setsRTR666020FRNase-Free Centrifuge Tubes (1.5 mL)50 EART Self prepared reagents: β- Mercaptoethanol, anhydrous ethanol (newly opened or dedicated for RNA extraction).Preparation and important precautions before the experimentTo prevent RNase pollution, attention should be paid to the following aspects:1) Use RNase free plastic products and gun heads to avoid cross contamination.2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3) Prepare the solution using water without RNase.4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.2. The extracted samples should avoid repeated freeze-thaw cycles, otherwise it will affect the quantity and quality of RNA extraction.3. Before use, please check if there is any crystallization or precipitation in the Buffer RL. It can be heated at 56 ℃ and re solved. Please add Buffer RL before use β- Mercaptoethanol, with a final concentration of 1%. Add 10 to 1ml Buffer RL µ L β- Mercaptoethanol. join β- The buffer RL room temperature of mercaptoethanol can be stored for one month.4. Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.5. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.6. If downstream experiments are highly sensitive to DNA, it is recommended to treat RNA with DNase I that does not contain RNase.Operation steps1. Sample processing1a organization: Grind the organization in liquid nitrogen. Add 600 to every 20-30 mg of tissue µ L Buffer RL (check if it is added before use) β- Mercaptoethanol), tissue sample less than 20 mg plus 350 µ Buffer RL. The sample volume shall not exceed one tenth of the buffer RL volume.1b Single layer culture of cells: The cells are directly lysed or processed into cell suspensions in a culture bottle, centrifuged to obtain cell precipitates, and the supernatant is discarded. 600 is added every 6-10 cm2 of culture area µ Buffer RL, less than 6 cm2, add 350 µ Blow buffer RL several times to fully crack it.1c cell suspension: Centrifuge at 12000 rpm (~13400 × g) for 1 minute to discard the supernatant and obtain cell precipitate. Add 600 cells every 5 × 106-1 × 107 cells µ Buffer RL, less than 5 × 106 cells added to 350 µ Blow buffer RL several times to fully crack it.Attention:1) Try to eliminate the cell culture medium as much as possible, as it may inhibit cell lysis and affect RNA production.2) Try to fully suspend and lyse the cells, otherwise it will affect RNA production.2. After the sample is fully lysed, it should be left at room temperature for 5 minutes to completely separate the protein nucleic acid complex.3. Centrifuge at 2000rpm for 2-5 minutes, take the supernatant and proceed to the next step.4. Add 1 volume (600) µ L or 350 µ l) Mix 70% ethanol (prepared without RNase water) well.Attention: Adding ethanol may cause precipitation and will not affect subsequent experiments.5. Add all the solution obtained in step 4 to the Spin Columns RM that has been loaded into the collection tube. If it is not possible to add all the solution to the adsorption column at once, please transfer it in two batches, centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube. Attention: The maximum loading capacity of the adsorption column is 100 µ g, do not overload, otherwise it will affect the yield and purity of RNA.6. Add 700 to the adsorption column µ Centrifuge at 12000 rpm for 1 minute, discard the waste liquid from the collection tube, and place the adsorption column in the collection tube.Optional steps: If conducting RNA experiments that are highly sensitive to trace amounts of DNA, replace step 6 with the following steps.1) Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 15 seconds, discard the waste liquid, and place the adsorption column back into the recovery manifold.2) Preparation of DNase I mixture: Take 52 µ Add 8 RNase Free Water to it µ 10 x Reaction Buffer and 20 µ DNase I (1 U/ µ l) Mix well and prepare to a final volume of 80 µ The reaction solution of L.Attention: The above system is configured according to our company's DNase I reaction system. Please refer to the corresponding manual for other company products.3) Add 80 µ l of the prepared DNase I reaction solution directly to the adsorption column and incubate at 20-30 ℃ for 15 minutes.4) Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 15 seconds, discard the waste liquid, and place the adsorption column back into the recovery manifold.7. Add 500 to the adsorption column µ Buffer RW2 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.8. Repeat step 7. 9. Centrifuge at 12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry.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 in a new RNase free centrifuge tube, and add 30-50 to the middle of the adsorption column in the air µ Place RNase Free Water at room temperature for 1 minute, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store RNA at -70 ℃ to prevent degradation.Attention:1) The volume of RNase Free Water should not be less than 30 µ l. Small volume affects the recovery rate.2) If you want to increase RNA production, you can use 30-50 µ Repeat step 10 for the new RNase Free Water.3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column and repeat step 10... Read More |