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 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