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Cat. No.: IKPAG-20 (for 20T)

Cat. No.: IKPAG-100 (for 100T)

Description

Immunoprecipitation Kit with Protein A+G Agarose Gel is a kit used for immunoprecipitation or co-immunoprecipitation of target proteins or protein complexes. It utilizes high-quality recombinant Protein A+G agarose gel in combination with user-provided specific antibodies. The immunoprecipitated products obtained using this kit can be used for the detection of target proteins or components of protein complexes.

This kit includes high-quality Protein A+G agarose gel and optimized and validated reagents necessary for immunoprecipitation (IP) experiments, making the immunoprecipitation or co-immunoprecipitation (Co-IP) experiments simple, convenient, and efficient. When used in conjunction with specific antibodies, it is widely used for immunoprecipitation, co-immunoprecipitation, or purification of target proteins or protein complexes.

Immunoprecipitation or co-immunoprecipitation is a common experimental technique used to study protein-protein interactions (PPIs). It involves using specific antibodies and a medium that can bind to the antibodies, such as Protein A/G agarose or Protein A/G magnetic beads, or directly using a medium coupled with specific antibodies, such as agarose gel or magnetic beads. Antigen-antibody complexes are then separated from the solution by centrifugation or magnetic force, allowing the target protein to be separated from complex samples. The isolated proteins can be further analyzed using techniques such as Western blotting or mass spectrometry.

Protein A is a cell wall surface protein found in Staphylococcus aureus with a molecular weight of 42 kDa, while Protein G is an immunoglobulin-binding protein expressed by C or G type streptococcal bacteria. Protein A and Protein G have similar functions and can specifically bind to mammalian immunoglobulins (Ig), typically in the Fc region of immunoglobulins. However, some studies have shown that Protein A can also bind to the Fab region of human VH3 family, and Protein G may also bind to the Fab region to some extent. Additionally, their binding capabilities to different subclasses of immunoglobulins vary. Recombinant modified Protein A and G can be coupled to agarose gel in a specific manner, making them suitable for immunoprecipitation or antibody purification.

Protein A+G agarose gel is suitable for immunoprecipitation of all antibodies that can be immunoprecipitated by Protein A agarose gel or Protein G agarose gel individually. This includes human IgG1, IgG2, IgG3, IgG4; mouse IgG1, IgG2a, IgG2b, IgG3; rat IgG1, IgG2a, IgG2b, IgG2c; as well as rabbit, goat, and other polyclonal antibodies.

The recombinant Protein A and Protein G in this product can specifically bind to the Fc region of most mammalian IgG antibodies. Their molecular weights are 14 kDa and 22 kDa, respectively. Through modification, these recombinant Protein A and Protein G retain only the amino acid sequences relevant to binding with IgG Fc region, while removing sequences that may cause nonspecific binding outside the binding sites. This effectively reduces nonspecific binding. Each molecule of Protein A and Protein G in this product can bind to 2 and 3 IgG molecules, respectively.

This kit includes high-quality Protein A+G agarose gel, Normal Mouse IgG, Normal Rabbit IgG (serving as negative controls), and optimized buffers such as Lysis Buffer, TBS (10X), Protease Inhibitor Cocktail (100X), Acid Elution Buffer, Neutralization Buffer, SDS-PAGE Sample Loading Buffer (2X), and other necessary reagents for immunoprecipitation. These components make the immunoprecipitation or co-immunoprecipitation experiments simple, convenient, and efficient. The immunoprecipitation procedure using this kit is depicted in Figure 1. After appropriate washing, the Protein A+G agarose gel is incubated with a specific antibody, and Protein A+G binds specifically to the Fc region of the antibody. After a certain incubation period, a Protein A+G agarose gel-antibody mixture (beads-Ab complex) is formed. Then, the sample is added, and the antigen in the sample is recognized specifically by the Fab region of the antibody, resulting in the formation of a Protein A+G agarose gel-antibody-antigen immunocomplex (beads-Ab-Ag complex). The immunocomplex is washed to remove unbound proteins, and then the bound immunocomplex is eluted from the agarose gel using acid elution buffer or SDS-PAGE sample loading buffer for subsequent detection.

Features

• Strong specificity and high binding capacity to target proteins. The Protein A+G Agarose Gel provided in this kit is a 25% gel suspension, prepared by mixing Protein A and Protein G agarose gel in a 1:1 ratio. Each milliliter of Protein A+G agarose beads (sediment) contains approximately 10 mg of recombinant Protein A and 1 mg of Protein G, capable of binding to over 25 mg of human IgG. The binding is highly specific, with minimal nonspecific binding to non-target proteins.
• Reusable for multiple times, cost-effective. Under normal conditions, this product can be reused for 3-5 purifications using the same antibody. However, if used for co-immunoprecipitation to detect protein-protein interactions, reuse is not recommended.

• Characteristics:    Description
• Product content:    25% settled gel in proper buffer
• Beads structure:    4% cross-linked agarose
• Average beads size:    90μm
• Coupled protein:    Recombinant Protein G
• M.W. of protein:    14kDa Protein A and 22kDa Protein G
• Ligand concentration:    10mg Protein A and 1mg Protein G per ml settled gel
• Binding capacity:    ≥25mg human IgG per ml settled gel
• Elution method:    Elution with acid, competing peptide or SDS‐PAGE loading buffer
• Application:    Suitable for IP, Co-IP, Protein purification
• Storage:    -20°C

This kit provides two elution methods. Based on the structure, biological function, and requirements for subsequent applications of the target protein in the antibody complex, this kit offers two elution methods: acidic solution and SDS-PAGE sample loading buffer. If there is a corresponding competitive peptide available, it can also be used for elution.

The Protein A+G Agarose Gel provided in this kit is a 25% gel suspension, and the packaging volume represents the total volume. Each milliliter contains 0.25 ml of pure gel (precipitate). For routine immunoprecipitation experiments, 20 μl of gel suspension is recommended for every 100 μl of sample. The small package and medium package of this kit allow for 20 and 100 immunoprecipitation experiments, respectively, with corresponding negative control experiments conducted 4 and 20 times, respectively.

Precautions

• If the target protein involved in immunoprecipitation is phosphorylated or acetylated, it is recommended to prepare the corresponding phosphatase inhibitor and deacetylase inhibitor.
• The provided Lysis Buffer in this kit has been extensively tested and is suitable for sample lysis and subsequent washing in many immunoprecipitation or co-immunoprecipitation experiments. However, due to the complexity and specificity of protein samples in immunoprecipitation or co-immunoprecipitation, this Lysis Buffer may not be suitable for all sample lysis and washing steps. If the performance of the provided Lysis Buffer is unsatisfactory, it is recommended to explore and adjust the lysis and washing solutions on your own.
• Before using the Agarose Gel, it should be thoroughly resuspended by inverting several times to ensure uniform mixing.
• The Agarose Gel contains 50% glycerol and trace amounts of preservatives. Before use, it is advisable to wash the gel three times with an appropriate solution such as TBS to eliminate interference from glycerol and preservatives.
• During immunoprecipitation, it is recommended to prepare a working solution of normal IgG from the same species as the primary antibody, at the same dilution ratio or final concentration, for the removal of non-specific binding or as a negative control. The kit provides an adequate amount of Normal Mouse IgG and Normal Rabbit IgG.
• After collecting protein samples, it is advisable to complete the purification process as soon as possible and always store them at 4°C or on ice to slow down protein degradation or denaturation.
• If centrifugation does not completely remove insoluble substances from the protein sample, the sample solution can be filtered through a 0.45 μm filter membrane.
• This product is for scientific research purposes by professionals only. It is not intended for clinical diagnosis or treatment, and should not be used for food or drug purposes. It should not be stored in residential areas.
• For your safety and health, please wear lab attire and disposable gloves when handling.

Storage

Store at -20°C, valid for one year.

Only for research and not intended for treatment of humans or animals

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