Electrophoresis Flashcards
What are the building blocks of proteins?
Amino acid residues
Describe gel electrophoresis
- Gel cast in a thin slab shape with wells
- Immersed within a buffer
- Proteins separated within a gel in a series of pores
What are the different detection methods used for electrophoresis?
Fluorescent stain
Silver stain
Protein staining (in situ)
- coomassive brilliant blue dyes used as 0.1% (w/v) in
methanol, distilled H2O + acetic acid (9:9:2)
What is the significance of the buffer in gel electrophoresis?
Provides ions to carry the current
Maintains a relatively constant pH
The pH of the solution and nature of amino acids R groups have significant effect on protein migration
In basic conditions, what is the overall protein charge?
net -ve charge due to COO- carboxyl group
What is the distance migrated by proteins in electrophoresis determined by?
Migration of protein not determined by intrinsic electric charge but by weight
What is the role of SDS-PAGE’s strong anionic detergent?
Solubilises dissociate and denature most proteins to single polypeptide chains
Disrupts H+ bonds
Blocks hydrophobic interactions
- binds at ratio of 1.4g of SDSg-1 of protien conferring net negative charge to polypeptide in proportion to length
What are the different types of electrophoretic gel?
- Agarose
2. Polyacrylamide
Which amino acid group determines the charge of the amino acid?
R group determines if the amino acid is neutral, acidic or basic
The protein backbone has no charge
What is protein electrophoresis?
Migration of any protein in an electric field depending on pI and pH
What is a discontinuous buffer system?
Uses different buffers
- > non restrictive large pore gel
- > resolving gel - greater resolution
- > have different buffers for stacking gel ,resolving gel, and tank buffer
What is the pI of proteins?
pI is constant for any given protein
What is electrophoresis?
The migration of charged macromolecules in an electric field
What is the significance of pH in protein electrophoresis?
pH of solution determines the charge of protein
When is Native non denaturing gel electrophoresis?
Used when native conformational need analysing
What is SDS-PAGE?
Most commonly used electrophoretic technique for separation containing disulfide bond cleavage agents e.g. β-mercaptoethanol
How is Agarose gel formed?
- polysaccharide extract from seaweed
- prepared by dissolving powdered agarose in buffer
- heated and poured into casting tray
- polymerased when cooled
Outline the main features of native gel electrophoresis
- Biological activity of proteins preserved (not denatured)
- Polyacrylamide / agarose used
- Runs without SDS
- Proteins separated by size, charge and shape
- Separates proteins even with same molecular weight
What is electrophoresis migration based on?
- Current
- Size
- Shape
- Charge
- Resistance
What is the use of agarose gel?
Has a relatively low resolving power as pores larger
used to separate large proteins >200KDa at a [0.5-2%]
Outline the features of Denaturing SDS-PAGE gel electrophoresis
- Negatively charged, strong anionic detergent SDS
- SDS denatures and dissociates proteins into polypeptide chains
- SDS complexes with proteins to mask charge
- Separtion based on molecular size only
What is a continuous buffer system?
Buffer system using same buffer in gel, the sample and tank
What is the net charge of proteins in acidic buffer conditions
net +ve charge due to NH3+
Describe Hb electrophoresis
pH range of 8-9 (alkaline)
majority proteins will be negatively charged
What are the features of Native gel electrophoresis?
Run without SDS
Proteins aren’t denatured
Separation based on charge:Size ratio and shape
Charge changes with buffer pH changes
How are the separated proteins detected in SPEP?
A densitometer is used to scan separated proteins in the gel pattern shows protein fractions
What is the use of electrophoresis?
Useful for separating/purifying macromolecules consisting of many sub units with multiple ionisable charged groups
What are the 2 major protein groups in blood serum?
- Albumin
- Globulins
What are the 2 apparatus types of electrophoresis?
- Horizontal - usually for agarose gel
2. Vertical - for polyacrylamide gel
What do we have to ensure when choosing type of electrophoresis apparatus?
- Electric field is uniform across gel
- To cool to prevent thermal artefacts
- Is access to gel monitoring and loading
What is the pI?
pI - isoelectric point
Zwitterions, when protein has no net charge at a certain pH neutral due to COO- and NH3+
What is blood composed of?
Blood composed of cells and plasma made up of water, proteins, salts, glucose, hormones and clotting factors
What is the polyacrylamide gel’s pore size determined by?
Polyacrylamide has smaller pores than agarose determined by [polyacrylamide]
What are buffer systems classified as?
- Continuous
- Discontinuous
What is the charge of proteins?
Proteins can either have a net -ve or +ve charge dependent on the pH of the buffer
What are the benefits of native gel electrophoresis?
- Can separate proteins with identical molecular weight
- Proteins can be recovered in native state
- Can steady binding
How is polyacrylamide gel formed?
Formed from synthetic small molecule acrylamide
- polymerises into long chains in presence of APS & TEMED
APS = catalyst
TEMED = initiator
What are the clinical applications of native gel electrophoresis?
Can measure specific proteins in blood
SPEP (serum protein electrophoresis) uses an electric field to separate proteins into groups of similar size, shape and charge helps identify disease
What is the significance of [gel]?
[gel] determines effective separation range of SDS-PAGE
SDS-PAGE not suitable for small peptides of molecular weight <10KDa
- dis/continuous buffer system can be used in protein gel electrophoresis
Describe the macromolecules in electrophoretic gel
In an electric field, negatively charged molecules migrate towards positive pole
What are the types of gel used in native electrophoresis?
Agarose & Polyacrylamide
What is the role of buffers?
Supplies current carrying ions in electrophoretic cell
Maintains desired pH
Provides a medium for heat dissipation
Describe the structure of amino acids
2 ionisable groups
- carboxyl group terminus
- amino group terminus
Variable side chain (R group)
What is the most common technique for protein electrophoresis?
Vertical slab gel electrophoresis (polyacrylamide)
