Important Notes
1. This product is for research use only and restricted to scientific research by qualified professionals.
2. Please read this instruction manual carefully before performing protein–protein interaction assays.
3. Unless otherwise stated, all procedures are recommended to be performed at 4°C to minimize protein degradation. If cell lysis is incomplete, sonication may be applied after treatment with lysis buffer.
4. Anti-V5 Nanobody magnetic beads should be stored in storage solution to prevent drying. Mix thoroughly by gentle inversion several times before use. Vigorous vortexing is not recommended to avoid antibody denaturation.
5. Boiled beads lose their binding capacity and should not be reused.
6. Reagent volumes may be adjusted according to actual experimental conditions. Preliminary experiments are recommended to verify immunoprecipitation or optimize lysis conditions.
7. Please observe safety precautions and comply with standard laboratory reagent handling protocols.
8. The lysis buffer included in this kit already contains protease inhibitors. Other appropriate inhibitor cocktails may be used for specific requirements.
9. Protein samples should be purified promptly after collection and kept at 4°C or on ice to reduce protein degradation or denaturation.
10. Positive and negative controls are recommended for immunoprecipitation or purification experiments.
Procedure (Unless otherwise specified, all procedures should be per-formed at 4°C)
1 Reagent Preparation
Additional Required Materials:
(1) Reagents to be prepared by the user:
a) Primary Antibody: V5 tag antibody.
b) Secondary Antibodies: Goat Anti-Mouse IgG H&L (HRP), Goat Anti-Rabbit IgG H&L (HRP).
c) Other Reagents: TBST, Electrophoresis Buffer, Transfer Buffer, Reducing SDS-PAGE Gel.
(2) Required Equipment:
Electrophoresis Apparatus, Transfer Apparatus, Imaging System
The above reagents, if required, can be ordered from Aladdin: V5 tag Mouse mAb (Ab133823), Recombinant V5 tag Antibody (Ab086830), Goat Anti-Rabbit IgG H&L (HRP)(Ab176443), Goat Anti-Mouse IgG H&L (HRP)(Ab179001) and Goat Anti-Rabbit IgG H&L (HRP)(Ab170144).
2 Solution Preparation
You may use the buffers provided in the kit, or prepare different buffer systems according to actual needs. It is recommended to filter all buffers through a 0.22 μm or 0.45 μm membrane filter before use. Buffers should be stored at 4°C. If any reagent appears cloudy, discard it immediately.
(1) Prepare an appropriate amount of inhibitor-containing lysis buffer based on the proportion of using 100−200 μL of inhibitor-containing lysis buffer for lysis and 300–600 μL of inhibitor-containing lysis buffer for washing per 0.5-1 million cells. Mix Lysis Buffer and Protease Inhibitor Cocktail (100×) at a ratio of 100:1. For example, add 10 μL of Protease Inhibitor Cocktail (100×) to 1 ml of Lysis Buffer to obtain 1 ml of inhibitor-containing lysis buffer (Lysis Buffer with Protease Inhibitor Cocktail). The prepared inhibitor-containing lysis buffer should be placed on ice or at 4°C.
Note: The inhibitor-containing lysis buffer should be prepared fresh before use and should not be frozen and stored for later applications.
(2) reparation of 10×Wash Buffer: Dilute the 10×wash buffer with deionized water at a 9:1 ratio. For example, add 9 mL of deionized water to 1 mL of 10×wash buffer, and mix well to obtain the 1×wash buffer.
(3) Beads Washing: Gently resuspend the Anti-V5 Magnetic Beads to form a homogeneous gel suspension. Typically, use 20 µL of the well-mixed gel suspension per 250 μg (the following immunoprecipitation steps are described based on adding 20 μL of gel suspension per sample). Transfer an appropriate amount of Anti-V5 Magnetic Beads into a clean centrifuge tube, and add 1×wash buffer to a final volume of approximately 0.5 mL. Allow to stand on the magnetic rack for 1 minute and discard the supernatant. Repeat the above steps twice.
Note: Using wide-bore tips (e.g., by cutting off the tip end with scissors) may facilitate pipetting of the gel suspension.
3 Preparation of Test Samples (Note: Perform all sample lysis steps at 4°C or on ice)
(1) For the preparation of serum samples:
If the target protein is abundant, dilute the serum sample with Lysis Buffer to a final target protein concentration of 50-150 µg/mL. Keep the diluted sample on ice for immediate use or store at -20°C for long-term preservation.
(2) For Adherent Cell Lysis and Preparation:
Aspirate the culture medium and wash the cells twice with PBS. Remove all residual liquid completely. Add 100-200 µL of Lysis Buffer with inhibitors per 0.5-1 million cells (equivalent to one well of a 6-well plate). Pipette gently to ensure thorough contact between the lysis buffer and cells. Animal cells are typically lysed within 1-2 seconds of contact with the buffer. For plant cells, lyse on ice for 2-10 minutes. After complete lysis, use a cell scraper to detach the cells and transfer the lysate to a 1.5 mL microcentrifuge tube. Centrifuge at 10,000-14,000×g for 3-5 minutes at 4°C. Collect the supernatant for protein concentration determination before proceeding to subsequent immunoprecipitation or co-immunoprecipitation experiments.
Note: A small amount of insoluble material, primarily genomic DNA, may be present after lysis and will form a pellet upon centrifugation.
(3) For Suspension Cell Lysis and Preparation:
Collect cells by centrifugation at 250-1,000×g for 5 minutes at room temperature. Wash the pellet twice with PBS and remove all residual liquid completely. Gently vortex or tap the tube to disperse the cells. Add 100-200 µL of Lysis Buffer with inhibitors per 0.5-1 million cells. Mix and incubate on ice for 5-20 minutes (mix several times during incubation). Tap the tube or pipette gently to ensure complete cell lysis; no significant cell pellet should remain after thorough lysis. If processing number of cells, aliquot them into tubes containing 0.5-1 million cells per tube before lysis. Large cell clumps are more difficult to lyse completely, whereas smaller numbers of cells allow better contact with the lysis buffer and lyse more efficiently. After complete lysis, centrifuge at 10,000-14,000×g for 3-5 minutes at 4°C. Collect the supernatant for protein concentration determination before subsequent immunoprecipitation or co-immunoprecipitation experiments.
Note: A small amount of insoluble material, primarily genomic DNA, may be present after lysis and will form a pellet upon centrifugation.
(4) For Bacterial or Yeast Sample Lysis and Preparation:
For 1 mL of bacterial or yeast culture, centrifuge to pellet the cells and remove the supernatant. Wash the pellet twice with PBS and remove all residual liquid completely. Gently vortex or tap the tube to resuspend and disperse the bacterial or yeast cells. Add 100-200 µL of Lysis Buffer with inhibitors. Gently vortex or tap the tube to mix, and lyse on ice for 2-10 minutes. For improved lysis efficiency, bacteria and yeast can be pretreated with lysozyme and lyticase, respectively, before adding the Lysis Buffer with inhibitors. After complete lysis, centrifuge at 10,000-14,000×g for 3-5 minutes at 4°C. Collect the supernatant for protein concentration determination before subsequent immunoprecipitation or co-immunoprecipitation experiments.
Note: A small amount of insoluble material, primarily genomic DNA, is likely present after lysis and will form a pellet upon centrifugation.
(5) For Tissue Sample Lysis and Preparation:
Mince the tissue into small fragments. Add approximately 100-200 µL of Lysis Buffer per 20 mg of tissue. Homogenize the mixture using a glass homogenizer or other suitable homogenization device. Thorough homogenization ensures complete tissue lysis. After lysis, centrifuge at 12,000×g for 5 minutes at 4°C. Collect the supernatant for protein concentration determination before subsequent immunoprecipitation or co-immunoprecipitation experiments.
Note: A small amount of insoluble material, primarily genomic DNA, is likely present after lysis and will form a pellet upon centrifugation.
4 Immunoprecipitation (IP)
(1) Beads Addition and Incubation
Add magnetic beads to the protein sample at a ratio of 20 μL bead suspension per 250 μg of protein. Incubate the mixture on a rocking platform or rotary mixer for 2 hours at room temperature or overnight at 4°C.
(2) Separation Using a Magnetic Rack
After incubation, place the tube on a magnetic rack and let it stand for 1 minute. Transfer the supernatant to a new microcentrifuge tube and save it for potential future analysis.
(3) Washing
Add 0.5 mL of Wash Buffer and resuspend the beads by gently pipetting up and down. Place the tube on the magnetic rack and let it stand for 1 minute. Discard the supernatant. Repeat the washing process three times using Wash Buffer. The protein-beads complexes are now obtained.
Note: The completeness of washing can also be monitored by measuring the OD280 of the wash solution. If the OD280 reading is greater than 0.05, increase the number of washes appropriately.
5. Protein Elution
Elution with SDS-PAGE Loading Buffer: For every 20 μL of original bead volume, add 30 μL of wash to resuspend the beads, followed by 30 μL of 2×SDS Loading Buffer. Mix gently by flicking the tube, then heat at 95-100°C for 5-10 minutes. Centrifuge at 5000×g for 1 minute. Collect the supernatant for SDS-PAGE electrophoresis or Western Blot analysis.
Note 1: Since Anti-V5 nanobody magnetic beads are free from antibody heavy/light chain contamination, the denaturing elution method is preferred for higher elution efficiency.
Note 2: The recommended loading volume for WB is 20 μL or less. The remaining sample can be stored at -20°C.
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