The His-tag Immunoprecipitation Kit is designed for efficient co-precipitation of tagged bait proteins and target proteins. The kit contains high-quality His-tag agarose magnetic beads along with optimized and validated essential reagents for immunoprecipitation, making Immunoprecipitation (IP, also known as Pull-down) or Co-Immunoprecipitation (Co-IP) experiments simpler, more convenient, and more efficient. It is widely used for immunoprecipitation, co-immunoprecipitation, or purification of His-tagged fusion proteins or their protein complexes.
This product contains high-quality His-tagged protein agarose magnetic beads (IDA-Ni), suitable for purifying histidine-tagged (6xHis-tagged) proteins from various expression sources, such as E. coli, yeast, insect cells, and mammalian cells. Based on magnetic agarose bead microspheres, the beads are chemically coupled with a tridentate iminodiacetic acid (IDA) ligand. After chelation with nickel ions (Ni²⁺), a relatively stable backbone structure is formed, providing more sites for coordination with the imidazole rings on the histidine tag, thereby effectively binding the target protein. The kit offers advantages including simple operation, strong binding capacity, and broad applicability.
The Ni IDA Magarose Beads provided in this kit is a 50% gel suspension, with the packaging volume referring to the total volume. Each milliliter contains 0.5 ml of pure gel (precipitate). For routine immunoprecipitation experiments, use 20 μl of gel suspension per 250 μg of sample.
| H1506147 | Components | 10T | 40T | Storage Temperature | Quantity Per Test | | H1506147A | Ni IDA Magarose Beads | 0.25 mL | 0.8 mL | 4°C | 20 µL per 250 μg sample | | H1506147B | 1 × Lysis Buffer | 10 mL | 40 mL | 4°C | 150 µL per 250 μg sample | | H1506147C | His Peptide
| 0.1 mL | 0.4 mL | -20 °C | 1.5 µL per 100 μL wash Buffer | | H1506147D | Acid Elution Buffer | 1 mL | 4 mL | 4°C | 100 µL per 250 μL sample | | H1506147E | 10x Wash Buffer
| 20 mL | 80 mL | 4°C | 500 µL per 250 μg sample | | H1506147F | Neutralization Buffer
| 0.1 mL | 0.4 mL | 4°C | 10 µL per 100 μL Acid Elution Buffer
| | H1506147G | Protease Inhibitor Cocktail(100 × ) | 0.1 mL | 0.4 mL | -20 °C | 1.5 µL per 150 μL Lysis buffer | | H1506147H | 2 × SDS-PAGE Loading buffer | 0.25 mL | 0.8 mL | -20 °C | 20 µL per 250 μg sample |
Note: For conventional immunoprecipitation experiments, use 20 μl of chromatography media per 250 μg of sample. |
Precautions
1. The amount of Ni IDA Magarose Beads used can be adjusted according to the actual experimental needs.
2. Before performing protein - protein interaction experiments, please read this manual carefully.
3. Unless otherwise specified, all procedures are recommended to be carried out at 4°C to minimize potential protein degradation. If cell lysis is incomplete, sonication may be used after applying the lysis buffer.
4. The beads should be stored in storage solution to prevent drying. Mix thoroughly before use.
5. Beads that have been boiled lose their binding capacity and should not be reused.
6. This product is intended for research use by qualified professionals only.
7. Please observe safety precautions and follow laboratory reagent handling protocols.
8. The lysis buffer included in this kit already contains protease inhibitors. If specific requirements exist, other appropriate inhibitor cocktails may be considered.
9. Before use, Ni IDA Magarose Beads must be fully resuspended by inverting several times to ensure a homogeneous mixture.
10. After collection, protein samples should be purified as soon as possible and kept at 4°C or on ice at all times to slow down protein degradation or denaturation.
Procedure
1. Reagent Preparation
The following reagents and materials that may be required for the experiment are not provided and need to be prepared separately:
(1) Reagents to be prepared by the user:
a) Primary Antibody: His 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: His tag (C-terminal) Mouse mAb ( Ab155835 )、Recombinant His tag Antibody ( Ab086830 )、Goat Anti-Rabbit IgG H&L (HRP) ( Ab176443)、 Recombinant Protein A/G, HRP conjugate(rp303272)、Goat Anti-Mouse IgG H&L (HRP) (Ab179001)
2. Solution Preparation
uffering agents provided in the kit can be used, or alternatively, different buffer systems can be prepared according to actual requirements. It is recommended that all buffers be filtered through a 0.22 μm or 0.45 μm membrane prior to use. Buffers should be stored at 4°C. Discard immediately if the reagent appears turbid.
a) 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 per 0.5–1 million cells. Mix Lysis Buffer and Protease Inhibitor Cocktail (100x) at a ratio of 100:1. For example, add 10 μl of Protease Inhibitor Cocktail (100x) 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.
b) Preparation of His Peptide Elution Buffer (10 mg/mL): Dilute a certain amount of the His peptide stock solution with wash buffer to a final concentration of 150 μg/mL. For instance, add 1.5 μL of the peptide to 98.5 μL of wash buffer and mix well to achieve a final concentration of 150 μg/mL. Adjustments can be made.
c) Preparation of 10x Wash Buffer: Dilute the 10x wash buffer with deionized water at a 9:1 ratio. For example, add 9 mL of deionized water to 1 mL of 10x wash buffer, and mix well to obtain the 1x wash buffer.
d) Beads Washing: Gently resuspend the Ni IDA Magarose 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 Ni IDA Magarose Beads into a clean centrifuge tube, and add 1x 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)
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
a) 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.
b) 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.
c) 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 a large number of cells, it is recommended to 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.
d) 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.
e) 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)
a) 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.
b) 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.
c) 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
Baseds on the characteristics of the tagged protein and the requirements of downstream experiments, choose one of the following three elution methods.
a) Non-Denaturing Elution
For every 20 μL of the original bead volume, add 100 μL of His‑peptide elution buffer. Incubate the mixture on a vertical rotator at 4 °C for 1–2 h, then place it on a magnetic stand for 1 minute. Aspirate the supernatant to collect the eluted fraction, which contains the His‑tagged protein and its complexes. To improve elution efficiency, the incubation time may be extended or elution may be repeated. The His‑tagged protein and its complexes can be stored at 4 °C for immediate use, or at –20 °C/–80 °C for long‑term storage. If elution is insufficient, the His‑peptide concentration can be increased or multiple elution steps can be performed. Once the beads are fully captured, aspirate the supernatant into a new centrifuge tube for Western Blot analysis.
b) Elution with SDS-PAGE Loading Buffer
For every 20 μL of original bead volume, add 30 μL of PBS (user to provide) to resuspend the beads, followed by 30 μL of 2xSDS Loading Buffer. Mix gently by flicking the tube, then heat at 95-100°C for 5-10 minutes. Centrifuge at 1000 rpm for 1 minute. Collect the supernatant for SDS-PAGE electrophoresis or Western Blot analysis.
Note: The recommended loading volume for WB is 20 μL or less. The remaining sample can be stored at -20°C.
c) Acidic Elution
For every 20 μL of original bead volume, add 100 μL of Acid Elution Buffer. Mix thoroughly and incubate on a rocking platform or rotary mixer for 5 minutes at room temperature. Centrifuge at 1000 rpm for 1 minute and transfer the supernatant to a new microcentrifuge tube. If neutralization is required, add 10 μL of Neutralization Buffer to adjust the pH to neutral.
Note: The efficiency of acidic elution may vary depending on the specific target protein. If high elution efficiency is critical, the pH of the Acid Elution Buffer can be optimized within the range of pH 2.5-3.1. Accordingly, the pH or volume of the Neutralization Buffer should also be adjusted. Specific conditions need to be optimized by the use.
