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
