Lecture 7 Protein Purification and Identification Flashcards
what does protein purification do?
It isolates the target protein from other cellular components.
Why is Protein Purification Important?
helps understand the structure and function of a protein.
what is the main challenge in protein purification?
Since, cells contain a complex mixture of proteins, DNA, molecules, and organelles, the challenge is to isolate the desired protein.
what are the methods for protein isolation? SCSS
Size sorting through gel filtration
Charge sorting ion exchange
Shape sorting
Sifting through centrifugation
What are the different methods for breaking open a cell? MOC
Mechanical methods, Osmotic pressure and Chemical methods
What are the mechanical methods used to break open a cell? GSV
Grinding, sonication, vortexing
What is the osmotic pressure method for breaking open a cell?
Placing cells in a hypotonic solution
What is the chemical method used to break open a cell?
Detergents or chemicals to dissolve membranes
Why is it important to choose the right cell type and what is an example to help understand this?
Different cells express different proteins. For example, pancreatic beta cells express insulin.
what are the different cellular compartments? NMC
Nucleus, mitochondria, and cytosol
What are the methods for isolating cell compartments? NMC
Nuclear fraction, Mitochondrial fraction, Cytosolic fraction
what is a method to quantify proteins?
Spectrophotometry
what is the equation of Spectrophotometry?
A = ε × c × l
what is the purpose of Spectrophotometry?
measures protein absorbance
what are the factors that chromatology considers to separate proteins?
based on size, charge, or binding interactions.
how does Size Exclusion Chromatography work? What does it use?
Separates proteins by size using porous beads.
how are large and small proteins affected in Size Exclusion Chromatography?
Large proteins pass quickly
small ones get trapped
how does Ion Exchange Chromatography work? What does it use?
Separates proteins by charge using a charged resin.
how does Affinity Chromatography work?
Separates proteins based on specific binding interactions (e.g., lock and key mechanism).
what is cation exchange in Ion Exchange Chromatography?
binds positive proteins
what is anion exchange in Ion Exchange Chromatography?
binds negative proteins
what is an example of Affinity Chromatography?
Nickel NTA column binds histidine residues.
how does Dialysis work?
Purifies proteins by removing small molecules through a semipermeable membrane.
how does High-Pressure Liquid Chromatography (HPLC) work?
Separates proteins using pressure and their hydrophobicity.
what method is used to assess protein purity?
SDS-PAGE
what are the breaking and reformings of non-covalent bonds like?
Weak bonds constantly breaking and reforming
how does SDS-PAGE work?
separates proteins based on size
what type of chromatology are non-covalent bonds important for?
affinity chromatography
