| Description | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | 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 | Inquire | M666110 Component 96 T Storage M666110A Buffer WSL 40 mL RT M666110B Buffer MSL 40 mL RT M666110C Buffer CW1 (concentrate) 90 mL RT M666110D Buffer GW1 (concentrate) 40 mL RT M666110E Buffer GW2 (concentrate) 50 mL RT M666110F Buffer EB 30 mL RT M666110G Proteinase K 4×1.25 mL RT M666110H M666110 Component 96 T Storage M666110A Buffer WSL 40 mL RT M666110B Buffer MSL 40 mL RT M666110C Buffer CW1 (concentrate) 90 mL RT M666110D Buffer GW1 (concentrate) 40 mL RT M666110E Buffer GW2 (concentrate) 50 mL RT M666110F Buffer EB 30 mL RT M666110G Proteinase K 4×1.25 mL RT M666110H Magbeads V3 2×1 mL RTProduct Introduction：The reagent kit provides a simple, fast, and efficient method for extracting genomic DNA from blood samples. In the presence of high salt, DNA binds to the surface of silica coated Magheads. After rinsing, high-purity DNA is eluted in Buffer EB or deionized water. The purified DNA has good purity (A260/280 ratio between 1.7-1.9) and high integrity (>15 kb), and can be used for downstream experiments such as second-generation sequencing, quantitative PCR, and chip detection.Self provided instruments and reagents1) Constant temperature mixer2) 2/15 ml magnetic frame3) 32 channel nucleic acid extractor4) 96 channel nucleic acid extractor5) 96 DW Plate6) 8 channel Comb7) Spin tips pack8) Anhydrous ethanolPreparation and important precautions before the experiment1.Before the first use, add anhydrous ethanol to Buffer CW1, Buffer GW1, and Buffer GW2 according to the label of the reagent bottle and mark them properly.2.Magheads are strictly prohibited from freezing or centrifugation. Freezing and centrifugation may cause irreversible damage to Magheads.Operation stepsI. Manual single tube operation1. Use punching forceps to take 1 blood spot with a diameter of 6 mm or 4 blood spots with a diameter of 3 mm (depending on the actual situation) from the blood spot and place them in a 2.0 mL centrifuge tube.2. Add 40 to the centrifuge tube µ L Protein K and 300 µ L Buffer WSL, then place the centrifuge tube on a constant temperature mixer at 75 ℃ and 1200 rpm, shake and crack for 45 minutes to form Lysate. Remove the centrifuge tube from the constant temperature mixer, centrifuge briefly, and take the supernatant.Attention: If there is no constant temperature mixer, vortex the centrifuge tube for 10 seconds and incubate it in a 75 ℃ water bath for 30 minutes. During this period, vortex every 10 minutes for 10 seconds.3. Suck the supernatant into a new 2.0 mL centrifuge tube and add 300 µ L Buffer MSL, 300 µ L isopropanol and 20 µ L Magheads V3. Afterwards, place the centrifuge tube on a constant temperature mixer at 25 ℃ and 1600 rpm, shake and crack for 15 minutes, or invert the centrifuge tube and mix continuously for 15 minutes.4. Place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, discard the solution thoroughly (keep the centrifuge tube fixed on the magnetic stand).5. Remove the centrifuge tube from the magnetic frame and add 900 µ L Buffer CW1 (please check if anhydrous ethanol has been added before use), vortex point shake for 1 minute or vortex shake for 5 seconds, and then place it on a constant temperature mixer at 25 ℃ and 1600 rpm to shake and mix for 2 minutes (ensure that Magheads are in a mixed state during the shaking process). Afterwards, place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, gently invert the magnetic stand and wash the impurities on the centrifuge tube cover to completely discard the solution (keep the centrifuge tube fixed on the magnetic stand).6. Remove the centrifuge tube from the magnetic frame and add 500 µ L Buffer GW1 (please check if anhydrous ethanol has been added before use), vortex point shake for 1 minute or vortex shake for 5 seconds, and then place it on a constant temperature mixer at 25 ℃ and 1600 rpm to shake and mix for 2 minutes (ensure that Magheads are in a mixed state during the shaking process). Afterwards, place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, gently invert the magnetic stand and wash the impurities on the centrifuge tube cover to completely discard the solution (keep the centrifuge tube fixed on the magnetic stand).7. Remove the centrifuge tube from the magnetic frame and add 900 µ L Buffer GW2 (please check if anhydrous ethanol has been added before use), vortex point shake for 1 minute or vortex shake for 5 seconds, then place it on a constant temperature mixer at 25 ℃ and 1600 rpm, shake and mix for 2 minutes (ensure that Magheads are in a mixed state during the shaking process). Afterwards, place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, gently invert the magnetic stand and wash the impurities on the centrifuge tube cover to completely discard the solution (keep the centrifuge tube fixed on the magnetic stand).8. Remove the centrifuge tube from the magnetic frame and add 300 µ After shaking with 75% ethanol for 1 minute or 5 seconds, place the mixture on a constant temperature mixer at 25 ℃ and 1600 rpm for 2 minutes (ensure that the Magheads are in a mixed state during the shaking process). Afterwards, place the centrifuge tube on a magnetic stand and let it stand for 1 minute. After Magheads are completely adsorbed on the side wall of the centrifuge tube, gently invert the magnetic stand and wash the impurities on the centrifuge tube cover to completely discard the solution (keep the centrifuge tube fixed on the magnetic stand).9. Keep the centrifuge tube fixed on the magnetic frame, use a pipette to further remove the solution from the bottom and cover of the centrifuge tube, and then leave it at room temperature for 5-10 minutes to allow the ethanol to evaporate completely.10. Remove the centrifuge tube from the magnetic frame and add 50-200 µ L Buffer EB. Vortex oscillation causes the magnetic beads to completely suspend in the eluent and then place them on a constant temperature mixer at 56 ℃ and 1600 rpm for 10 minutes of shaking and elution, or incubate the centrifuge tube in a 56 ℃ water bath for 10 minutes, with vortex oscillation every 3 minutes for 10 seconds.11. Place the centrifuge tube on a magnetic stand and let it stand for 2 minutes. After Magheads are completely adsorbed on the side wall of the centrifuge tube, transfer the eluent to a new centrifuge tube using a pipette and store at -20 ℃ for later use.II. Matching with CWE21001. Use punching forceps to take 1 blood spot with a diameter of 6 mm or 4 blood spots with a diameter of 3 mm (depending on the actual situation) from the blood spot and place them in a 2.0 mL centrifuge tube.2. Add 40 to the centrifuge tube µ L Protein K and 300 µ L Buffer WSL, then place the centrifuge tube on a constant temperature mixer at 75 ℃ and 1200 rpm, shake and crack for 45 minutes to form Lysate.3. Add the corresponding reagents to the 96DW deep well plate according to the table below. Position Reagent 1&7 Colume Lysate: All Buffer MSL: 300 µL isopropanol：300 µL Magbeads V3: 20 µL 2&8 Colume Buffer CW1: 900 µL 3&9 Colume Buffer GW1: 500 µL 4& 10 Colume Buffer GW2: 900 µL 5& 11 Colume 75%ethanol: 300 µL 6& 12 Colume Buffer EB: 70 µL4.Place the deep well plate and magnetic sleeve that have been added to the reagent at the corresponding positions of CWE2100/CWE3200, run the blood slide extraction program, and after about 40 minutes, the program ends. Remove the deep well plate and magnetic sleeve.5.Transfer the elution products from columns 6 and 12 of the deep well plate to a 1.5 mL centrifuge tube for low-temperature storage.III. Matching with CWE9601. Use punching forceps to take 1 blood spot with a diameter of 6 mm or 4 blood spots with a diameter of 3 mm (depending on the actual situation) from the blood spot and place them in a 2.0 mL centrifuge tube.2. Add 40 to the centrifuge tube µ L Protein K and 300 µ L Buffer WSL, then place the centrifuge tube on a constant temperature mixer at 75 ℃ and 1200 rpm, shake and crack for 45 minutes to form Lysate.3. Add the corresponding reagents to the 96DW deep well plate according to the table below Position Reagent Plate 1 Lysate: All Buffer MSL: 300 µL isopropanol ：300 µL Magbeads V3: 20 µL Plate 2 Buffer CW1: 900 µL Plate 3 Buffer GW1: 500 µL Plate 4 Buffer GW2: 900 µL Plate 5 75% ethanol : 300 µL Plate 6 Buffer EB: 70 µL4. Place the deep well plate and magnetic sleeve that have been added to the reagent at the corresponding positions on CWE960, run the blood slide extraction program, and after about 40 minutes, the program ends. Remove the deep well plate and magnetic sleeve.5. Transfer the elution products from Plate 6 to a 1.5 mL centrifuge tube for low-temperature storage... Read More | Cell viability and cytotoxicity assays are usually used for drug screening and compound cytotoxicity testing. The CCK-8 kit uses highly water-soluble tetrazolium salt ( called WST-8 ) to produce water-soluble WST-8 for cell proliferation and cytotoxicity assays. Unlike MTT, WST-8 and WST-8 have no Cell viability and cytotoxicity assays are usually used for drug screening and compound cytotoxicity testing. The CCK-8 kit uses highly water-soluble tetrazolium salt ( called WST-8 ) to produce water-soluble WST-8 for cell proliferation and cytotoxicity assays. Unlike MTT, WST-8 and WST-8 have no cytotoxicity in cell culture medium, so multiple downstream experiments can be performed using the same detection plate. CCK-8 method is a convenient colorimetric method for the determination of cell viability. It does not need the solubilization process and only needs the least steps to provide the results. The CCK-8 method can be used for the determination of 96-well microplates and high-throughput screening of 384-well microplates. Advantage：At present, the commercially available liquid CCK-8 kits generally have defects such as harsh storage conditions ( -4C or -20 ), unstable use in different pH ranges, and easy deterioration ( discoloration or precipitation ). The solid instant CCK-8 kit adopts a new formula and Swiss process, which overcomes these shortcomings of the liquid CCK-8 kit. It can be stored at room temperature for a long time ( > 3 years ), ready to use, stable in a wide pH range, and the experimental results are more reliable. Compared with the liquid CCK-8 kit, the solid-soluble CCK-8 kit has higher sensitivity and the biological response time is shortened by half.Application scope:It can be used for drug screening, cell proliferation assay, cytotoxicity assay, tumor drug sensitivity test and activity detection of biological factors. Operating instructions:This reagent kit can be used for drug screening, cell proliferation assay, cytotoxicity assay, tumor drug sensitivity assay, and activity detection of biological factors.1. Carefully and slowly tear along the gap in the packaging bag;2. Pour all the powder in the bag into a clean container containing 10mL of ultrapure water, shake continuously for 1 minute, and use it when the solid is completely dissolved;3. Unused reagents must be stored at low temperatures below 4 ℃.Equipment required for testing:Enzyme reader 96 well plate with 450-490 nm filter;Carbon dioxide incubator;96 well plate, sterilized transparent plate for cell detection;Multi channel pipette (8 or 12 channels: 10-100 µ l);Blood cell counter or cell counter.Cell viability testing:1. Inoculate cell suspension (100 µ l/well) into a 96 well plate and pre culture the plate in a carbon dioxide incubator for 24 hours (37 ℃, 5% CO2);2. Add 10 µ l of CCK-8 solution to each well (be careful not to generate bubbles in the well as it may affect the reading of OD value);3. Incubate the culture plate in the incubator for 1-4 hours;4. Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader;5. If the OD value is not determined temporarily, 10 µ l of 0.1M HCI solution or 1% w/v SDS solution can be added to each well, and the culture plate can be covered and stored in the dark at room temperature. Within 24 hours of measurement, the absorbance will not change.Cell proliferation toxicity testing:1. Inoculate cell suspension (100 µ l/well) into a 96 well plate and pre culture the plate in an incubator for 24 hours (37 ℃, 5% CO2);2. Add 10ul of different concentrations of the substance to be tested to the culture plate;3. Incubate the culture plate in the incubator for an appropriate period of time (e.g. 6, 12, 24, or 48 hours);4. Add 10 µ l of CCK-8 solution to each well (be careful not to generate bubbles in the well as they may affect the reading of the OD value);5. Incubate the culture plate in the incubator for 1-4 hours;6. Measure the absorbance at 450nm using an enzyme-linked immunosorbent assay (ELISA) reader;7. If the OD value is not determined temporarily, 10 µ l of 0.1M HCI solution or 1% w/v SDS solution can be added to each well, and the culture plate can be covered and stored in the dark at room temperature. Within 24 hours of measurement, the absorbance will not change.Calculation method for cell survival rate/inhibition rate:Cell survival rate=[As Ab)/(Ac Ab)] x 100%Inhibition rate=[(Ac As)/(Ac Ab)] x 100%As: absorbance of experimental wells (including cells, culture medium, CCK-8 solution, and drug solution);Ac: absorbance of control wells (including cells, culture medium, CCK-8 solution, without drugs);Ab: Blank well absorbance (including culture medium and CCK-8 solution, excluding cells and drugs).Points for attention： 1.Unused reagents must be stored at low temperature below 4 °C, and stored in the dark at-20 °C for two years after unpacking, so as to avoid repeated thawing ; 2.The culture time of CCK-8 is generally 1-4 hours, but the naked eye can be taken out to observe the color degree in about 30 minutes. According to the cell type, the conditions need to be explored. The best reaction time of CCK-8 is based on the best time of specific color development.3. It is recommended to do a few holes to explore the number of inoculated cells and the culture time after adding CCK-8 reagent ; 3.The WST-8 in this kit will react with reducing agents ( such as some antioxidants ) to interfere with the detection. Before the cell proliferation-toxicity test, the background OD can be checked to confirm whether there is a reducing agent in the substance to be tested. If the effect of reducing agent needs to be removed, the fresh medium can be replaced before adding CCK-8 ( remove the medium, wash the cells twice with the medium, and then add the new medium ) ; 4.Phenol red in the medium does not affect the experimental results, and the absorbance of phenol red can be eliminated by deducting the absorbance of the background in the blank hole during calculation, so it will not affect the detection. 5.It is recommended to use a multi-channel pipette to reduce the difference between parallel holes. When adding CCK-8 reagent, it is recommended to add it obliquely to the wall of the culture plate, not to insert it under the liquid surface of the medium, which is easy to produce bubbles and interfere with OD determination. 6.If the drug contains metal, it has an effect on the color of CCK-8. The final concentration of 1mM lead chloride, ferric chloride and copper sulfate will inhibit the color reaction of 5 %, 15 % and 90 %, and reduce the sensitivity. If the final concentration is 10mM, the color reaction will be 100 % inhibited ; 7.When using a 96-well plate for detection, if the cell culture time is long, attention should be paid to the evaporation problem. On the one hand, because a circle around the 96-well plate is the easiest to evaporate, the method of discarding the surrounding circle can be adopted, and the same amount of PBS, water or culture medium can be added. On the other hand, the 96-well plate can be placed near the water source in the incubator to alleviate evaporation ; 8.When using standard 96-well plates, the minimum inoculation amount of adherent cells is at least 1,000 cells / well ( 100µl medium ). The sensitivity of detecting white blood cells is relatively low, so it is recommended that the inoculation amount should not be less than 2,500 cells / well ( 100 µl medium ). If you want to use a 24-well plate or a 6-well plate experiment, first calculate the corresponding inoculation amount per well, and add the CCK-8 solution according to 10 % of the total volume of the medium per well ; 9.Cell culture time varies according to the type and number of cells ( per well ), usually the color of white blood cells is weak, requiring a longer culture time ( 4 hours ) and a large number of cells ( ~ 105 cells / well ) ; 10.CCK-8 reagent is very low toxic to cells. The continuous reaction between it and dehydrogenase in living cells makes the color of the solution deepen and the OD value increase. The following methods can terminate the CCK-8 reaction ( 96-well plate ) : a ) After the color reaction, the culture plate was placed in a refrigerator at 4 ° C ; b ) 10µL 0.1MHCL solution was added to each well ; c ) 10 µL 1 % ( w / v ) SDS ( sodium dodecyl sulfate ) solution was added to each well. After the reaction stopped, the OD value should be measured within 24 hours. 11.To determine the specific number of cells, it is recommended to do the standard curve at the same time... Read More |