Bacic Immunoprecipitation(免疫沉淀实验)
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实验概要
Immunoprecipitation (IP) is one of the most widely used immunochemical techniques. Immunoprecipitation followed by SDS-PAGE and immunoblotting, is routinely used in a variety of applications: to determine the molecular weights of protein antigens, to study protein/protein interactions, to determine specific enzymatic activity, to monitor protein post-translational modifications and to determine the presence and quantity of proteins. The IP technique also enables the detection of rare proteins which otherwise would be difficult to detect since they can be concentrated up to 10,000-fold by immunoprecipitation.
In the IP method, the protein from the cell or tissue homogenate is precipitated in an appropriate lysis buffer by means of an immune complex which includes the antigen (protein), primary antibody and Protein A-, G-, or L-agarose conjugate or a secondary antibody-agarose conjugate. The choice of agarose conjugate depends on the species origin and isotype of the primary antibody. The methods described are comparable and the choice of method depends on the specific antigen-antibody system.
Immunoprecipitation (IP) is one of the most widely used immunochemical techniques. Immunoprecipitation followed by SDS-PAGE and immunoblotting, is routinely used in a variety of applications: to determine the molecular weights of protein antigens, to study protein/protein interactions, to determine specific enzymatic activity, to monitor protein post-translational modifications and to determine the presence and quantity of proteins. The IP technique also enables the detection of rare proteins which otherwise would be difficult to detect since they can be concentrated up to 10,000-fold by immunoprecipitation.
In the IP method, the protein from the cell or tissue homogenate is precipitated in an appropriate lysis buffer by means of an immune complex which includes the antigen (protein), primary antibody and Protein A-, G-, or L-agarose conjugate or a secondary antibody-agarose conjugate. The choice of agarose conjugate depends on the species origin and isotype of the primary antibody. The methods described are comparable and the choice of method depends on the specific antigen-antibody system.
主要
试剂
Agarose conjugate: Protein A immobilized on Sepharose® CL-4B [Details：Product No. P3391] or Protein G-Agarose 4B [Details：Product No. P3296] or Protein L-Agarose [Details：Product No. P3351], or antibody-agarose conjugate [Details：secondary antibody conjugates according to the species origin and isotype of the primary antibody]
Primary antibody for immunoprecipitation [Details：refer to the product's specifications] and non-relevant antibody[Details：negative control]
HNTG buffer: 20 mM HEPES buffer pH 7.5 [Details：Product No. H4034], containing 150 mM NaCl [Details：Product No. S9625], 0.1% (w/v) Triton X-100 [Details：Product No. X100] and 10% (w/v) glycerol [Details：Product No. G9012]
Washing buffer [Details：Ice cold] : HNTG buffer, or PBS pH 7.4 [Details：Product No. P4417], or other buffers (RIPA buffer) according to the level of washing stringency required
Laemmli sample buffer [Details：e.g. 1x, 2x (Product No. S3401) or 3x ] with or without 2-mercaptoethanol (2-ME) [Details：Product No. M7154]
Agarose conjugate: Protein A immobilized on Sepharose® CL-4B [Details：Product No. P3391] or Protein G-Agarose 4B [Details：Product No. P3296] or Protein L-Agarose [Details：Product No. P3351], or antibody-agarose conjugate [Details：secondary antibody conjugates according to the species origin and isotype of the primary antibody]
Primary antibody for immunoprecipitation [Details：refer to the product's specifications] and non-relevant antibody[Details：negative control]
HNTG buffer: 20 mM HEPES buffer pH 7.5 [Details：Product No. H4034], containing 150 mM NaCl [Details：Product No. S9625], 0.1% (w/v) Triton X-100 [Details：Product No. X100] and 10% (w/v) glycerol [Details：Product No. G9012]
Washing buffer [Details：Ice cold] : HNTG buffer, or PBS pH 7.4 [Details：Product No. P4417], or other buffers (RIPA buffer) according to the level of washing stringency required
Laemmli sample buffer [Details：e.g. 1x, 2x (Product No. S3401) or 3x ] with or without 2-mercaptoethanol (2-ME) [Details：Product No. M7154]
