Please enable JavaScript.
Coggle requires JavaScript to display documents.
Protein Purification - Coggle Diagram
Protein Purification
SDS-PAGE
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
Separates proteins based on size
Need to denature proteins
Principle
SDS has negative charge
2-mercaptoethanol or DTT reduces disulphide bonds
Heat to 95\(^o\) for 5 min
Results in unfolded protein coated in negative charge
Protein samples are loaded into gel + electric current is applied
-ve charged proteins migrate towards positive electrode
Proteins move through mesh of polyacrylamide according to size
Smaller proteins will move further through the gel than larger ones
Visualised using Coomassie blue
Mr marker used on gel
Used during protein purification to check purity of a sample
Factors affecting protein migration
Generally migrate based on mass
Can migrate at a different size
Large post-translational modifications (PTMs) cause proteins to move at higher mass
e.g. ubiquitylation and glycosylation
Small PTMs don't affect protein migration
e.g. phosphorylation
Proteins that are not fully denatured may move as a complex
e.g. dimer
Inefficient reduction of disulphide bonds
High content of basic amino acids
Western Blotting
Uses a specific antibody to detect a protein of interest
After SDS-PAGE
Transfer proteins to a membrane
Primary Ab binds target protein
Secondary Ab with tag for detection binds 1\(^o\) Ab
Protein is visualised
Protein sources
Recombinant proteins
Produced in bacteria
Expression in
E.coli
Pros
Fast growth rate
Lots of protein-expression bacteria produced very quickly
Can transform bacteria with plasmid DNA very rapidly (<5mins)
Relatively cheap
Cons
Proteins may not fold correctly
High conc. protein can be insoluble (inclusion bodies)
Lack some post-translational modifications e.g. phosphorylation
General protocol
Transform BL21
E.coli
with plasmid
Pick a single colony + grow in 5ml of medium (37\(^o\)C 6-8hrs)
Transfer to 200ml medium (37\(^o\)C 16hrs)
Transfer to 1L medium (37\(^o\)C 1.5hrs)
Add 0.1-mM IPTG (37\^o\) 2-4hrs)
2 more items...
Protein purification from bacteria
Lyse cells without denaturing protein of interest
Freeze-thawing
Non-ionic detergent
Sonication (ultra high frequency sound)
Centrifuge again after lysis
Supernatant contains soluble cellular contents inc. proteins
Produced in other organisms
How?
Gene of interest is cloned into an expression plasmid/vector
Plasmid transferred into host cells (e.g. bacteria or human cells)
High levels of protein produced in host cells
Protein purified for functional studies
Plasmids/vectors
Restriction enzyme site
Antibiotic resistance gene
Promoter region
Endogenous proteins
From tissue
How?
Exploit a specific property of a protein to purify and enrich target protein
Size
Charge
Affinity tag
Hydrophobicity
Biological activity
From bacteria
After cell lysis and centrifugation
Purify protein from crude cell extract
Differential solubility
Often initial purification step
Techniques inc.
High salt conc.
Ammonium sulphate precipitation
High salt conc. leads to displacement of water molecules (salting out)
Water bonds with salt instead of proteins
Proteins bind each other and ppt. out
Ammonium sulphate is cheap, highly water-soluble, available at high purity + doesn't cause permanent denaturation of proteins
Different proteins have different solubilities in aqueous solution
Salt may need to be removed before next purification step
3 common methods of salt removal
Dialysis
4 more items...
Gel filtration chromatography
Diafiltration
Polyethylene glycol (PEG)
Heat denaturation
Altering pH
Precipitated protein (solid) can then be re-dissolved + purified further
Polar water molecules interact with hydrophilic regions of protein, increasing protein solubility
Anything that affects protein charge or protein-water interactions will affect solubility
Affinity chromatography
Size exclusion chromatography
Ion exchange chromatography
Hydrophobic interaction chromatography
Isoelectric focussing
Several rounds of purification may be required
Track protein throughout process
Western blotting (immunoblotting)
Assay (e.g. enzyme)
Differential solubility
Often initial purification step