| Description | This kit innovatively adopts a column-based purification method to rapidly, gently, and efficiently lyse animal tissues or cells for total protein extraction. It provides both denaturing and native lysis buffers, allowing users to select the appropriate option based on downstream application This kit innovatively adopts a column-based purification method to rapidly, gently, and efficiently lyse animal tissues or cells for total protein extraction. It provides both denaturing and native lysis buffers, allowing users to select the appropriate option based on downstream application requirements. The entire extraction process takes only 1–8 minutes. Thanks to the column purification technology, it can process sample-lysis buffer mixtures as small as 20 µL and up to 500 µL, yielding protein solutions with concentrations of 2–8 mg/mL while effectively preventing protein loss. The extracted proteins can be quantified using the BCA method (Cat. No.: R1491648/B665595).C1491674Component50TStorageC1491674ADenaturing Lysis Buffer25 mL2-8℃C1491674BNative Lysis Buffer25 mL2-8℃C1491674CPurification Columns50 unitsRT.C1491674DCollection Tubes50 unitsRT.C1491674EPlastic Grinding Pestles4 unitsRT.Key Features1.Simple and rapid operation: Denatured total proteins can be obtained in as little as 1 minute.2.No protein loss: Efficiently extracts DNA-binding proteins by disrupting DNA duplexes.3.Small sample volume, high yield: Processes mixtures as small as 20 µL, yielding protein concentrations of 2–8 mg/mL.4.Versatile applications: Includes two lysis buffers for extracting both denatured and native proteins.ProtocolI. Extraction of Denatured Total Protein1.Pre-chill the purification column and collection tube on ice.2.Sample processing: Add protease inhibitor cocktail to the denaturing lysis buffer at a 1:100 ratio shortly before use.2.1 Adherent cells:Wash cells with pre-chilled 1× PBS and aspirate the supernatant.Add the volume of denaturing lysis buffer specified in the appendix table to cover the culture surface, and pipette to mix.2.2 Suspension cells:Collect cells by low-speed centrifugation.Wash with pre-chilled 1× PBS, vortex, and centrifuge at 3,000 rpm for 2–3 minutes.Resuspend the cell pellet in PBS equal to the pellet volume.Add the specified volume of denaturing lysis buffer and vortex to lyse.Note: Partial incomplete lysis does not affect protein extraction. If the lysate is too viscous, directly transfer it to the purification column.2.3 Tissue samples:Place 15–20 mg of tissue on the purification column.Grind 50–60 times with a plastic pestle.Add 200 µL denaturing lysis buffer and grind another 30–60 times.Adjust lysis buffer volume proportionally for larger or smaller samples.Note: Reusable plastic pestles should be thoroughly rinsed with distilled water and dried.3.Centrifugation:3.1 Adherent or suspension cells: Transfer the lysate to the pre-chilled purification column and centrifuge at 14,000–16,000 rpm for 30 seconds.3.2 Tissue samples: Incubate the column at room temperature for 1–2 minutes, then centrifuge at 14,000–16,000 rpm for 1–2 minutes.4.Immediately place the collection tube on ice and discard the purification column. Denatured total protein extraction is complete.II. Extraction of Native Total Protein1.Pre-chill the native lysis buffer, purification column, and collection tube on ice.2.Sample processing: Add protease inhibitor cocktail to the native lysis buffer at a 1:100 ratio shortly before use.2.1 Adherent cells:Wash cells with pre-chilled 1× PBS and aspirate the supernatant.Add the specified volume of native lysis buffer and incubate on ice for 3–5 minutes. Pipette to mix.2.2 Suspension cells:Collect, wash, and resuspend cells as described in section I.Add native lysis buffer, vortex for 15 seconds, incubate on ice for 3–5 minutes, and vortex again for 10 seconds.2.3 Tissue samples:Grind tissue as described in section I, using native lysis buffer.3.Centrifugation:3.1 Adherent or suspension cells: Centrifuge at 14,000–16,000 rpm for 30 seconds.3.2 Tissue samples: Incubate on ice for 5 minutes (open lid), then close the lid and centrifuge at 4°C and 14,000–16,000 rpm for 1–2 minutes.4.Immediately place the collection tube on ice and discard the purification column. Native total protein extraction is complete.Cell Count (×10⁶)Lysis Buffer Volume (µL)0.3200.550110022003500 Appendix: Cell Number vs. Lysis Buffer VolumePrecautions1.High viscosity of the lysate is normal when using this kit.2.For safety, wear a lab coat and disposable gloves during operation.3.For research use only... Read More | Inquire | DescriptionCAR10 is a kit that contains a selection of 10 carbohydrates/sugars: Arabinose, Fructose, Galactose, Glucose, α-Lactose, Maltose, Mannose, Ribose, Sucrose and Xylose, which may be used for general research, as reagents or as reference compounds in analytical procedures | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Product Content R669990Component50 TStorageR669990ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669990B10×Reaction Buffer1 mL-20℃. Avoid freeze/thaw cycle.R669990CBuffer RL35 mLRTR669990DBuffer RW135 mLRTR669990EBuffer RW2 (concentrate)11 mLRTR669990FRNase-Free Water10 Product Content R669990Component50 TStorageR669990ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669990B10×Reaction Buffer1 mL-20℃. Avoid freeze/thaw cycle.R669990CBuffer RL35 mLRTR669990DBuffer RW135 mLRTR669990EBuffer RW2 (concentrate)11 mLRTR669990FRNase-Free Water10 mLRTR669990GSpin Columns RM with Collection Tubes50 setsRTR669990HRNase-Free Centrifuge Tubes (1.5 mL)50 EART ProductsThis kit combines highly efficient guanidine isothiocyanate cleavage technology with silica matrix membrane purification for the efficient extraction of total RNA from animal cells and tissues, typically up to 30 mg of tissue or 1x107 cells as a starting sample. The kit also allows recovery of incompletely purified RNA, in vitro transcription and RNA from enzymatic reactions. high quality RNA with molecular weights greater than 200 bases can be extracted and purified using the kit with virtually no DNA residue. If RNA experiments that are very sensitive to trace DNA are to be performed, residual DNA can be removed by on-column digestion using RNase-free DNase. The extracted RNA can be used in downstream experiments such as RT-PCR, Nothern Blot and Dot Blot. Self-contained reagents: β-mercaptoethanol, anhydrous ethanol (freshly opened or for RNA extraction).Pre-experiment Preparation and Important Notes1. To prevent RNase contamination, attention should be paid to the following aspects:1) Use RNase-free plastics and tips to avoid cross-contamination.2) RNase-free water should be used to prepare the solution.(3) Operators wear disposable masks and gloves, and change gloves diligently during the experiment.2. Avoid repeated freezing and thawing of the extracted samples, otherwise it will affect the amount and quality of RNA extraction.3. Please add β-mercaptoethanol to Buffer RL before use, add 10µl of β-mercaptoethanol to 1ml of Buffer RL. Buffer RL with β-mercaptoethanol can be stored for 1 month at room temperature.4. Anhydrous ethanol should be added to Buffer RW2 before first use according to the instructions on the reagent bottle label.5. Buffer RL may be heated at 56°C to dissolve if precipitation occurs and then left at room temperature.All centrifugation steps are performed at room temperature and all maneuvers are performed quickly.Procedure1. Sample handling1a Tissue: Grind tissue in liquid nitrogen. Add 600 µl Buffer RL for every 20-30 mg of tissue (check for addition of β-mercaptoethanol before use), and 350 µl Buffer RL for tissue samples of less than 20 mg. Sample volume is not to exceed one-tenth of the Buffer RL volume.1b Cells in monolayer culture: Lysed or processed into cell suspension directly in culture flask, centrifuged to obtain cell precipitate, discarded the supernatant, added 600µl Buffer RL for every 6-10 cm2 of culture area, 350µl Buffer RL for less than 6cm2, and blown several times repeatedly to make the cells lysed sufficiently.1c Cell suspension: centrifuge at 12,000 rpm (~13,400 × g) for 1 min and discard the supernatant to obtain the cell precipitate. Add 600 µl Buffer RL for every 5×106-1×107 cells, and 350 µl Buffer RL for less than 5×106 cells, and blow several times repeatedly to fully lysate.Note: 1) Try to get rid of the cell culture medium, which may inhibit cell lysis affecting RNA yield.2) Try to keep the cells well suspended and well lysed, otherwise RNA yield is affected.2. After the sample is fully lysed, leave it at room temperature for 5 minutes to allow complete separation of the protein-nucleic acid complex.3. Centrifuge at 12,000 rpm for 2-5 min and remove the supernatant for the following operations.4. Add 1x volume (600µl or 350µl) of 70% ethanol (prepared without RNase water) to the solution obtained in step 3 and mix well.Note: The addition of ethanol may produce a precipitate that will not affect subsequent experiments.5. Add all of the solution obtained in the previous step to the Spin Columns RM in the collection tube. If you cannot add all of the solution to the column at once, transfer it in two passes, centrifuge at 12,000 rpm for 1 minute, and discard the waste solution. Place the column back into the collection tube.Note: The maximum loading capacity of the adsorption column is 100µg, do not overload as this will affect the yield and purity of the RNA.6. Add 350 µl Buffer RW1 to the adsorbent column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid and put the adsorbent column back into the collection tube.7. Preparation of DNase I mixture: Take 52 µl of RNase-Free Water, add 8 µl of 10×Reaction Buffer and 20 µl of DNase I (1 U/µl) to it, mix well, and prepare a final volume of 80 µl of reaction solution.8. Add 80µl of DNase I mixture directly to the adsorption column and incubate at 20-30°C for 15 minutes.9. Add 200 µl Buffer RW1 to the adsorbent column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid and put the adsorbent column back into the collection tube.10. Add 500µl Buffer RW2 to the column (check that anhydrous ethanol is added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube, and put the column back into the collection tube.11. Repeat step 10.12. 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 a few minutes to thoroughly dry the anhydrous ethanol in the adsorption column.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.).13. Transfer the adsorbent column into a new centrifuge tube, add 30-50 µl of RNase-Free Water to the middle of the adsorbent membrane, leave it at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min, collect the RNA solution, and store the RNA at -70°C to prevent degradation.Note: 1) The volume of RNase-Free Wate should not be less than 30 µl, too small volume affects the recovery rate.2) If you want to increase the RNA yield, repeat step 13 with 30-50 µl of fresh RNase-Free Water.3) If the RNA concentration is to be increased, the resulting solution can be reintroduced into the adsorption column and step 13 repeated... Read More |