C6 Flashcards
Analytical method of protein interaction studies
- Protein array
- Sandwich immunoassay
- Yeasts 2 hybrid assay
- Immunoprecipitation
- Fluorescence resonance energy transfer (FRET)
Principle of protein array
Based on any ligand binding assay that relies on the formation of product with immobilised capture molecules & target molecules present in the solution
Characteristics of protein array
- Many types of substance (protein, antibody, aptamer)
- Spotted on array
- Equivalent to DNA microarray
Application of protein array
- Screening
- Test for protein-protein interactions
- Test for protein-drug interactions
- Test for enzyme-substrate interactions
Common label in protein array
- Cytochrome (Cy3)
- Cy5
- Biotin
- HRP
Procedure of protein array
- Sample obtained from cell, tissue
- Antibody array incubated with diluted Cy3/Cy5 labeled sample
- Array are scanned using microarray scanner
- Visualisation
- Analysis
Variation of protein array can be made
- Antibody chip to detect antigen
- Protein chip to detect interactions
- DNA chip to detect protein that bind DNA
- Ligand/receptor chip to detect signalling proteins
Principle of sandwich immunoassay
- Antibody with different specificity immobilised on surface
- Bind specific protein in sample
- Detect using second antibody
Pros & Cons of sandwich immunoassay
Pros
- Many different protein can be assay on the same chip
- Versatile
Cons
- Specificity difficult to verify
- Background variations
- Expensive
Principle of yeasts 2 hybrid assay
- Use to test if one protein (bait) interact with other protein (prey)
- Bait protein tagged to DNA binding domain of yeast GAL4 transcription factor
- A library of prey protein tagged to activation domain of GAL4
- DBD bind to reporter gene
Explain the interaction of protein in yeasts 2 hybrid assay
- Interaction between bait and prey bring DBD & AD of GAL4 together
- Induce transcription of reporter gene
- cDNA of prey gene cloned into second vector with AD
- Co-transforming yeasts cell with bait and prey vector so that the interaction of bait be studied
Principle of immunoprecipitation
- Capture multiprotein complex by using antibody to one of the protein in the complex
- Other members of the complex isolated & identify using MS, LC-MS-MS or western blot
- Result confirmed by doing second precipitation in the complex
Steps in immunoprecipitation
- Antibody specific to the target protein added to sample
- Antibody bind to specific protein
- Form antibody protein complex
- Secondary agent added to bind with Fc region of antibody
- Elution to remove immune complexes
Principle of pull down assay
- Similar to immunoprecipitation but use bait protein instead
- Other protein interact with bait will be captured
- Bait is tagged with ligand & immobilised on solid substrate
- Capture protein are eluted
- Analyse using MS
Principal of FRET
- Non radiative transfer of energy from excited fluorophore (donor) to acceptor fluorophore
- Only occur when molecules are in very close proximity (1-10nm)
