Key words: affinity chromatography protein

Key words: affinity chromatography protein protein interaction

Immunocoprecipitation and affinity chromatography:

Immunocoprecipitation is the most direct, classical and effective method to prove protein-protein interaction. For example, protein A can interact with B to form heterodimer, and both a and B can be precipitated by anti-A or anti-B antibody or agarose beads coupled with a or B affinity. Therefore, it is widely used in the study of protein interaction.

Common beads:

GST agarose (no antibody required)
ProteinA agarose
Basic methods:

  1. Express protein A

  2. Protein A binds to affinity beads

  3. To extract protein B from cell broken solution by using affinity beads with protein A

  4. Centrifugation and washing of affinity beads several times

  5. Treatment of affinity beads to desorb protein A and B (it can be boiled directly with buffer on SDS-PAGE)

Yeast two hybrid system

Basic principle, take the gal 4 system of S.cerevisiae as an example

Gal4 has two structurally separate and functionally independent domains.

N-terminal 1-147aa binds to DNA (BD).

C-terminal 768-881aa can activate transcription domain (AD).

BD + ad can activate the upstream activating sequence (UAS) of Gal4 effect gene.

The expression plasmids of N and C were constructed separately. For example, if the N-terminal of Gal4 is fused with the decoy protein (target protein A of the study), and the cell cDNA and C-terminal are fused, if the protein X encoded by cDNA can interact with a, then the C-terminal and N-terminal of Gal4 can be linked together, and then the gene expression downstream of UAS can be activated.

The galactosidase (Laz) gene was cloned to the downstream of URA3.

In the presence of X-gal, yeast forms blue (fields)

Gal4 gene deletion in yeast; Gal80 gene deletion (negative regulator of Gal4)

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