Methods In Protein Biochemistry Flashcards
Sonification
(Extract Protein)
Mechanically destroys the membrane
Produces heat
Must do in ice
Shearing
(Extract Protein)
Mechanically destroys the membrane
Mild Detergents
(Extract protein)
Dissolves the membrane
Biological Fractionation
(Separate/Purify)
Uses centrifugation to separate based on cell compartments
I.e. membrane, nucleus, organelles
Protein fractionation
(Separate/Purify)
uses centrifugation to separate based on density of protein
Size Exclusion Column Chromatography
(Separate/purify)
Separates based on size
Uses porous beads
Smaller proteins move slower
Ion Exchange Column Chromatography
(Separate/purify)
Separates based on charge
Uses stationary phase with negative or positive ligand
Positive ligand binds to negative proteins
Positive will flow through the column
Hydrophobicity Column Chromatography
(Separate/purify)
Separates based on hydrophobicity
Uses stationary phase with hydrophobic ligand
Hydrophobic molecules bind to stationary phase
Hydrophilic molecules flow through column
Affinity Column Chromatography
(Separate/purify)
Separates based on affinity to a ligand
Ligand will be fixed to stationary phase
Molecules with high affinity will bind to ligand
Other molecules will flow through column
SDS PAGE
(Evaluate purification)
Sample is run through polyacrylamide gel
SDS denatures/negatively charges proteins
Proteins migrate from negative to positive electrode
Based on molecular weight
Isoelectric Focusing
(Determine pI)
Sample is run thorugh a stable pH gradient
Non denaturing conditions
Proteins migrate thorugh the gradient based on charge
Negatively charged —> positive electrode
Positively charged —> negative electrode
When pH makes charge zero they stop migrating (pI)
Absorbance @ 280 nm
(Quantify)
Tryptophan and tyrosine must be present
Absorb light at 280 nm
Absorbance is proportional to concentration
Fast and easy ; accurate for single proteins
Other compounds absorbing at 280 shouldn’t be present
Pierce Assay
(Quantify)
Positively charge amino acids bind to a dye
Absorb light at 595 nm
Absorbance proportional to concentration
Useful for bulk proteins
Takes longer and requires standards and replicates
Mass Spectrometry
(Identify Unknown)
Fragments are separated and detected
Based on mass charge ratio
Mass and order of fragments = amino acid sequence
Amino acids —> protein identification
Circular Dichroism
(Determine structure)
Useful for determining presence of secondary structure
Measures molar absorption difference of left/right circularly polarized light
Does not provide detailed info on structure
X-ray Crystallography
(Determine structure)
Protein crystals exposed to x-rays
Diffraction pattern used to determine electron density
Protein model built on top by fitting amino acids
No size limits : big and small
Protein crystals are difficult to make
Cannot see protons and doesn’t work will with flexible regions
Models not representative of in vivo conditions
NMR Spectroscopy
(Determine structure)
Works for tertiary and secondary structure
Proteins in solution exposed to powerful magnetic fields
Nuclear spins are aligned
The way the spins return to original state is influence by neighboring atoms
Protein models can be built
Useful for flexible regions and if seen protons are required
Only for small proteins
Electron Tomography
(Determine structure)
Proteins frozen on grid and images are taken by electron microscope
Images averaged to produce model
Useful for membrane and fibrous proteins
Does no work well for flexible regions
Not representative of in vivo conditions
Homology Modeling
(Determine structure)
Using known structure of similar proteins
Structural hypothesis generated
Fast cheap and easy
Not the true structure
