Low Resolution Techniques Flashcards
Name low resolution techniques
For protein stability
- Thermal Shift Assay
- Thermal denaturation using Circular Dichroism
- SDS PAGE: purity & modifications
- Native Page: possible states
For structure determination
- Circular dichroism: secondary structures
- Dynamic Light Scattering: size, mw, shape
- Size exclusion chromatography coupled to Multi-Angle Light Scattering: oligomeric state, shape, mw, size
SDS Page
Objective: determine purity
Principle: Separate protein based on mass
Components:
-SDS: detergent & imparts - charge on protein
-B mercaptoethanol: reducing agent . Reduces disulfide bonds
-Heat: denatures protein
Circular Dichroism (CD) Spectrocopy
Objective:
1. For protein stability: monitor protein folding/unfolding
2. For protein structure: Identify 2 & 3 structures
Principle:
1. Monitor trp fluorescence at 280 nm
2. Determine alfa helices, beta sheers, randoms coils. Absorbs light at different degrees.
Components:
Circular Polarized light
Chiral molecules (optically active molecules)
Thermal Shift Assay
Objective: determine stability of protein as a function of increasinf temperature
Principle: External dye binds hydrophobic regions
Native Page
Objective: provides information about possible states of proteins (oligomers)
Principle: proteins separated based on mass/ shape / charge.Separate mixture of oligomeric proteins. Retain structures and interactions. Monomers travel faster
Componets
No reducing agent, detergent or heat
Steps
Isolate membrane & cytoplasm proteins
Run Native
Run SDS
Dynamic Light Scattering (DLS)
Objetive: determine size, shape, aggregates and purity
Principle: larger particles scatter light at a lower frequency due to slow motion
Brownian motion: frequency of scattered light is dependant on particle size
Size Exclusion Chromatography - Multiangle Light Scattering
(Sec Mals)
Objective: Separate proteins. Dtermine oligomeric state, shape, mw and size
Principle: Based on hydrodynamic radius & shape
