| Description | This kit enables convenient and efficient separation of nuclear and cytoplasmic proteins from animal cells or tissues. It employs a stepwise cell lysis protocol to isolate intact nuclei from the cytoplasm, followed by extraction of nuclear proteins.The kit contains potent detergents that extract notThis kit enables convenient and efficient separation of nuclear and cytoplasmic proteins from animal cells or tissues. It employs a stepwise cell lysis protocol to isolate intact nuclei from the cytoplasm, followed by extraction of nuclear proteins.The kit contains potent detergents that extract not only nuclear membrane proteins but also soluble proteins such as histones and nuclear transcription factors. Compared to traditional kits requiring 30–40 minutes for nuclear protein extraction, this product reduces the extraction time to 10 minutes. It delivers higher nuclear protein yields and superior nuclear-cytoplasmic separation, making it particularly suitable for Western Blot applications.N1491647Component50TStorageN1491647AWB nuclear/plasma reagent A20 mL2-8℃N1491647BWB nuclear/plasma reagent B0.5 mL2-8℃N1491647CWB nuclear/plasma reagent C5 mL2-8℃Key Features1.Rapid: Optimized protocol reduces extraction time by 10–20 minutes compared to traditional kits.2.High Yield: Formulated for Western Blot applications, delivering higher nuclear protein yields than conventional kits.3.High Purity: Excellent separation of nuclear/cytoplasmic proteins with minimal cross-contamination (outperforms traditional kits).4.Easy Operation: Simple protocol without ultracentrifugation gradients.ProtocolPlace all kit components on ice. Add 1× protease inhibitor (Cat. No. P665818) or 1× protease/phosphatase inhibitor (Cat. No. P752090) to Reagent A and Reagent C before use.Animal Cells1.Harvest Cells:Adherent cells: Discard medium, wash with PBS, scrape cells, and transfer to a tube. Centrifuge at 300 ×g for 5 min.Suspension cells: Centrifuge at 300 ×g for 5 min, wash with PBS, and repeat centrifugation.2.Resuspend cells in PBS, transfer 2 × 10⁶ cells to a 1.5 mL tube, and centrifuge at 300 ×g for 5 min. Discard supernatant.3.Add 200 µL Reagent A to the pellet, mix thoroughly, and incubate on ice for 10 min.4.Add 10 µL Reagent B, vortex at maximum speed for 5 sec, and incubate on ice for 1 min.5.Vortex again at maximum speed for 5 sec and incubate on ice for 2 min.Note: Adjust ice incubation time (1–3 min) based on cell type to avoid aggregation.6.Centrifuge at 1,600 ×g for 10 min (4°C). Carefully transfer the supernatant (cytoplasmic fraction) to a new tube. Store at -80°C if needed.Note: Avoid touching the pellet. Retain a minimal volume of supernatant to reduce contamination.7.Resuspend the pellet in 200 µL Reagent A, mix thoroughly, and centrifuge at 13,000 ×g for 5 min (4°C). Discard supernatant completely.Note: The pellet contains nuclei. Use a 10 µL tip to remove residual supernatant.8.Add 100 µL Reagent C to the pellet, vortex at maximum speed for 10 sec, and incubate on ice. Repeat vortexing every 2 min for 10 min.9.Centrifuge at 13,000 ×g for 10 min (4°C). Transfer the supernatant (nuclear protein fraction) to a new tube and store at -80°C.Animal Tissues1.Mince 20–80 mg tissue into small fragments in a 2 mL tube (optional: add PBS and grind with a syringe plunger). Centrifuge at 1,600 ×g for 3 min (4°C) to collect fragments.2.Add Reagent A (see Table 1 for volumes) to the fragments, transfer to a homogenizer, and homogenize on ice.Note: Cut pipette tips to facilitate transfer of tissue fragments.3.Transfer the homogenate to a pre-chilled tube and incubate on ice for 15 min.4.Add Reagent B (Table 1), vortex at maximum speed for 5 sec, and incubate on ice for 1 min.5.Vortex again for 5 sec and incubate on ice for 2 min.6.Centrifuge at 1,600 ×g for 10 min (4°C). Transfer the supernatant (cytoplasmic fraction) to a new tube. Store at -80°C if needed.7.Resuspend the pellet in the same volume of Reagent A as Step 2, mix, and centrifuge at 13,000 ×g for 5 min (4°C). Discard supernatant.8.Add Reagent C (Table 1) to the pellet, vortex at maximum speed for 10 sec, and incubate on ice. Vortex every 2 min for 10 min.9.Centrifuge at 13,000 ×g for 10 min (4°C). Transfer the supernatant (nuclear protein fraction) to a new tube and store at -80°C. Table 1. Recommended Reagent Volumes for Tissue Extraction组织重量/mgWB核/浆试剂A/µLWB核/浆试剂B/µLWB核/浆试剂C/µL20200101004040020200608004040080100050500Precautions1.Add 1× protease inhibitor (Cat. No. P665818) or 1× protease/phosphatase inhibitor (Cat. No. P752090) to Reagent A and Reagent C before use.2.This kit is optimized for Western Blot and is not compatible with SDS-sensitive applications.3.Quantify extracted proteins using the BCA Protein Assay Kit (Cat. No. R1491648/B665595).4.Wear a lab coat and disposable gloves for safety.5.For research use only... Read More | Inquire | Inquire | O665690 Component 50T Storage O665690A DNase I 1000 U -20℃.Avoid freeze/thaw cycle. O665690B 10×Reaction Buffer 1000 µL -20℃.Avoid freeze/thaw cycle. O665690C Buffer RLS 40 mL RT O665690D Buffer RW1 40 mL RT O665690E Buffer RW2 (concentrate) 11 mL RT O665690F RNase-Free Water O665690 Component 50T Storage O665690A DNase I 1000 U -20℃.Avoid freeze/thaw cycle. O665690B 10×Reaction Buffer 1000 µL -20℃.Avoid freeze/thaw cycle. O665690C Buffer RLS 40 mL RT O665690D Buffer RW1 40 mL RT O665690E Buffer RW2 (concentrate) 11 mL RT O665690F RNase-Free Water 10 mL RT O665690G Spin Columns FS with Collection Tubes 50 EA RT O665690H Spin Columns RM with Collection Tubes 50 EA RT O665690I RNase-Free Centrifuge Tubes (1.5 mL) 50 EA RTProduct IntroductionThis kit is suitable for extracting RNA from a wide range of plants, even from plants rich in polysaccharides and polyphenols, high quality RNA can be successfully extracted, such as rice leaves, wheat leaves, corn leaves, tobacco leaves, pine needles, ginkgo leaves, poplar leaves, pomegranate leaves, holly leaves, apples, peaches, pears, tomatoes, cherries, apricots, bananas, grapes, loquats, cinnamon rinds, cinnamon pulp, lychee fruit rinds, lychee pulp, soybean, peanut, corn, potato tuber, moonflower petal, pomegranate petal, shiitake mushroom, flat mushroom and other samples. The unique lysate formula can rapidly inactivate the RNA enzyme in the cell, effectively remove the effect of polysaccharide and polyphenol on RNA extraction, without the need for phenol, chloroform and other reagents, while using silicon matrix membrane adsorption of RNA for purification, the total RNA extracted is highly pure, without the contamination of genomes, proteins and other impurities, and can be used for Real Time RT-PCR, RT-PCR, It can be used for Real Time RT-PCR, RT-PCR, Northern Blot, Dot Blot, in vitro translation and other downstream experiments.RNA yieldSelf-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.(2) 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 rate and quality of RNA extraction.3. If Buffer RLS produces a precipitate, heat to dissolve it and leave at room temperature.4. Please add β-mercaptoethanol to Buffer RLS before use, add 20µl β-mercaptoethanol to 1ml Buffer RLS. Buffer RLS with β-mercaptoethanol can be stored for 1 month at room temperature.5. Anhydrous ethanol should be added according to the instructions on the reagent bottle label before using Buffer RW2 for the first time. Operation steps1. Homogenization: Take 50-100mg of plant tissue and quickly grind it into powder in liquid nitrogen, add 500µl of Buffer RLS (please check whether β-mercaptoethanol is added before use), and immediately mix it by vortexing with vigorous shaking.Note: For materials that are extremely rich in water content, such as watermelon pulp, tomato, pear pulp, etc., more material can be added appropriately, up to 200 mg; for starch-rich samples or mature leaves, the amount of Buffer RLS can be increased appropriately, up to 700 µl.2. Centrifuge at 12,000 rpm (~13,400 x g) for 2 min at 4°C.3. Transfer the supernatant into the filter columns (Spin Columns FS) that have been loaded into the collection tubes, centrifuge at 12,000 rpm at 4°C for 1 minute, carefully aspirate the supernatant in the collection tubes and transfer it to new RNase-Free centrifugation tubes (self-provided), avoiding the tip of the gun from touching the cell debris precipitation in the collection tubes as much as possible.4. Slowly add 0.5 times the volume of the supernatant in anhydrous ethanol, mix well (a precipitate may appear), and transfer the resulting solution together with the precipitate to a Spin Columns RM in a collection tube, or in two batches if you cannot add all of the solution at once. centrifuge the column for 1 minute at 12,000 rpm at 4°C. Dispose of the spent solution and place the column back into the collection tube. Centrifuge at 12,000 rpm for 1 minute at 4°C, discard the spent solution and return the column to the collection tube.5. Add 350 µl of Buffer RW1 to the adsorbent column RM, centrifuge at 12,000 rpm at 4°C for 1 min, discard the waste solution and put the adsorbent column back into the collection tube.6. Preparation of DNase I mixture: Take 52µl of RNase-Free Water, add 8µl of 10×Reaction Buffer and 20µl of DNase I (1U/µl) to it, mix well, and prepare a final volume of 80µl of reaction solution.7. Add 80µl of DNase I mixture directly to the adsorption column and incubate at 20-30°C for 15 minutes.8. Add 350 µl of Buffer RW1 to the adsorbent column RM, centrifuge at 12,000 rpm at 4°C for 1 min, discard the waste solution and put the adsorbent column back into the collection tube.9. Add 500 µl of Buffer RW2 to the adsorbent column RM (check that anhydrous ethanol is added before use), centrifuge at 12,000 rpm for 1 minute at 4°C, discard the waste solution and put the adsorbent column back into the collection tube.10. Repeat step 9.11. Centrifuge at 12,000 rpm for 2 minutes at 4°C.Note: The purpose of this step is to remove residual ethanol from the adsorption column; ethanol residue can interfere with subsequent enzymatic reactions (zymography, PCR, etc.).12. Load the adsorption column RM into new RNase-Free Centrifuge Tubes (1.5 ml), add 30-50 µl of RNase-Free Water dropwise to the middle part of the adsorption membrane overhang, leave it at room temperature for 2 min, and centrifuge at 12,000 rpm at 4°C for 1 min, and store the resulting RNA solution at -70°C to prevent degradation.Note: 1) The volume of RNase-Free Water 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 12 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 12 repeated... 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