L8-9: Purification of proteins Flashcards
What is heterologous expression?
The use of bacteria/yeast/mammalian/insect cells to make protein of interest which allows lots of it to be obtained but it isnt always in its native state
What are the biophysical properties of proteins?
Size
Mass
Shape/structure (primary to quaternary & folding)
Interactions
Redox potential
Charge
Hydrophobicity
Electromagnetic properties
What are the 2 main methods of separating proteins?
Chromatography
Gel electrophoresis
How are proteins isolated?
Using centrifugation :
Cells are homogenised then blended then filtered and centrifuged
What is centrifugal force?
A force appearing to act on an object when viewed in a rotating frame of reference
How can centrifugal force be expressed?
As relative centrifugal force:
RCF=centrifugal force/g
What are the 2 forces acting on a centrifuge?
Buoyant force and centrifugal force
What does buoyant force depend on?
The viscosity of liquid and density/size of particle
What type of centrifugation is needed for organelles and proteins?
Organelles - higher speeds
Proteins - ultra centrifugation
Which intrinsic biophysical properties have to be exploited after centrifugation?
Size
Mass
Shape
Interaction
Charge
Hydrophobicity
What are the principles of chromatography?
Sample of interest (proteins) have differential interaction strength between mobile and stationary phase
What are the steps of column chromatography?
1- protein mix in buffer added to column with stationary phase and suitable buffer
2- Buffer used as mobile phase and flows through column, proteins interacting with stationary phase move much slower than ones remaining in the mobile phase
3- Proteins in mobile phase exit the column first
What are the 2 different types of chromatography used?
Thin layer chromatography and column chromatography
How can the proteins be detected from the chromatography?
Using other biophysical properties
How can protein concentration be calculated in a chromatogram?
By integrating under the peak curve
What physical properties can proteins be separated by?
Affinity, Ion exchange, size exclusion, hydrophobic interaction, reversed phase and multimodal chromatography
How are proteins separated based on size exclusion?
Gel filtration, separates based on size (hydrodynamic radius), large separates first
How are proteins separated based on ion exchange?
Based on charge overall, charge distribution and charge density
What is the process of ion exchange?
Mix of proteins in buffer loaded into cation exchanger
+ive charged proteins adsorb to media displace Na+
Pulse of buffer is formed
pH decreases to aviod drastic ionic strength
Name examples of anion and cation exchangers
Anion:
Quaternary ammonium
Diethylaminoethyl
Diethylaminopropyl
Cation:
Sulfopropyl
Methyl sulfonate
Carboxymethyl
How are proteins separated based on hydrophobicity?
Differences in surface hydrophobicity, media modified with hydrophobic alkyl (-CH3)/aryl (-phenyl) groups, high salt concentration favours interaction of protein with media
(works like size exclusion chromatography)
What is the most commonly used salt for hydrophobicity?
(NH4)2SO4
How are proteins separated based affinity?
Stationary phase functionalised with chemical/protein that binds protein of interest
Name examples of chemicals that bind proteins of interest in affinity chromatography
ProteinA- IgG
Streptactin- Biotin or Strep-tag
NTA (nitriloacetic acid)- metals His-tag
Heparin- DNA binding proteins
What is IMAC? (chromatography)
Immobilised metal affinity chromatography
What is IMAC? (chromatography)
Required recombinant protein expression, His-tag (6-10 histidine residues added to protein), at N- or C- terminus, Histidine binds to metals like nickel and cobalt, produces pure protein quick
What is electrophoresis?
The movement of dispersed particles relative to a fluid under the influence of a spatially uniform electric field
What can electrophoresis separate?
Proteins, nucleic acids and glycans
What is gel electrophoresis?
When polymer gels act as molecular sieves
What are 2 different types of gels and what are they made of?
Agarose - linear polymer (polysaccharide)
Acrylamide - Organic compound with multiple functional groups
What are the factors that effect gel electrophoresis?
-Net charge of molecule
-Size of molecule
-Electric field strength (can denature)
-Properties of gel
-Properties of running buffer (pH, counter ions, Salts)
-Temperature
What are PAGE variants?
Polyacrylamide gel electrophoresis
What is native PAGE? (electrophoresis)
Separation of acidic proteins
Separation by charge/size
Samples added to buffer with dye/glycine/buffer
Good for looking at protein complexes
Post-translational modifications
What is SDS-PAGE?
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
What are the properties of SDS-PAGE?
Protein denatured with heat/SDS
SDS in gel/buffer: laemmli buffer
Formation of mixed proteins: SDS micelles
Charge by protein masked
Separation by mass
What proteins recalcitrant to denaturation?
Disulfides - require reducing agent
Thermostable proteins
What are the 2 discontinuous gels?
pH 6.5 stacking gel (focuses proteins before separation)
pH 8.8 resolving gel (separates proteins)
What are the buffer components in SDS-PAGE?
Glycine/chloride counter ions (migration)
SDS (denaturation)
Tris (pH 8.3)
What do gradient gels do in SDS-PAGE?
Increase concentration of acrylamide down gel
What buffers are used in agarose gel electrophoresis?
Tris pH 8.3
Acetate/Borate (counter ion)
EDTA (chelating agent)
What is the difference between agarose gel electrophoresis and PAGE?
Resolution isnt as high in agarose
How are gels visualised?
Using dyes
What are the properties of visualising gels?
They have different sensitivities and different visualisation conditions
How can molecular weight be estimated from gels?
-comparing migration of samples vs standard curve
-protein/nuc acid standards/markers run on gels
What is the calculation to find molecular weight using gels?
-measure migration dist of standards and dye front from top
-calculate relative motility standards (Rf)
-measure for unknowns
-plot log(MW) of standards vs Rf
-plot best fit and calculate eqn
-solve
What are common problems with gels? (anomalous gels)
Smileys (high voltage)
Melting gels (too hot)
Smearing (overloading)
Contaminants (double dipping)
Bubbles
Speckles
Floaty samples
Leaky gel tanks
What is absorption spectroscopy?
When a particular wavelength of light is absorbed
How is absorption spectroscopy affected by environment?
Bathochromic- shift to longer wavelength
Hypsochromic- shift to shorter wavelength
What are applications of UV/Vis absorption spectroscopy
Quantification of protein/nuc acids
Protein unfolding
Nuc acid unfolding
Characterisation of substrate/cofactor binding proteins
Biochemical assays
What is the Beer-Lambert Bouger Law
𝐴= 𝜀𝓁𝒸
𝜀- molar attenuation coefficient
𝓁- optical path length
𝒸- concentration of attenuating species
What are the key assumptions of the Beer-Lambert-Bouguer law?
Monochromatic/parallel illumination
Homogeneous solution
No scatter from medium
Linear absorbance change with conc
Linear response of detector
What are indirect measurements of quantifying proteins?
Lowry
Bradford
Biuret
BCA assay
