Electrophoresis Flashcards
What is the isoelectric point?
Where the protein has no net charge at that pH
What is the charge if pH
Net positive charge
What is the charge if pH>pI?
Net negative charge
What is pI?
Isoelectric point
What are the principles of electrophoresis?
Migration of charged particles in the electric field
What is electrophoresis useful for?
Separating/ Purifying macromolecules
What does migration of charges depend on in electrophoresis?
Charge, size or shape
What gel does the horizontal electrophoresis apparatus normally use?
Agarose
What gel does the vertical electrophoresis apparatus normally use?
Polyacrylamide
What is the function of the tank in electrophoresis?
Where the sample and buffer are attached to a power block
What is the function of the power block in electrophoresis?
Supplies electric current through the buffer
What is the function of the casting tray in electrophoresis?
Preparing the gel
What is the function of the sample comb in electrophoresis?
Makes an indentation in the gel that allows you to put your sample in the buffer before applying the current
What do you need to check when choosing apparatus (3)?
Uniform electric field across gel
Cooling to present thermal artefact
Access to gel loading an monitoring
Where is gel electrophoresis usually cast?
In a thin slab within wells
What does the buffer provide in gel electrophoresis (3)?
- ions to carry current
- relatively constant pH
- pH of solution and nature of R-groups have an important effect on migration of proteins
What is agarose made up of?
Polysaccharide extract from seaweed
How is agarose prepared?
Dissolving powdered agarose into buffer. Heat in microwave and pour into a casting tray.
Undergoes polymerisation when cooled
Why does agarose have a low resolving power?
Big pores
What is agarose used to separate (specifically what size)?
Large proteins >200kDa
What concentration is agarose used at?
0.5-2%
What is polyacrylamide formed from?
The small synthetic molecule acrylamide
What does acrylamide need to be in the presence of to polymerise?
Catalyst and initiator (APS and TEMED)
What is the pore size of acrylamide determined by?
Polyacrylamide concentration
What does TEMED stand for?
Tetra methyl ethylene diamine
What is the most popular technique for protein electrophoresis?
Vertical slab gel
What do buffers do in electrophoresis (3)?
Supply current carrying ions
Maintains desired pH
Provides medium for heat dissipation
What are the two classifications for buffers?
Continuous
Discontinuous
What is a continuous buffer?
Uses same buffer in gel, sample and tank
What is a discontinuous buffer (4)?
Different buffer
Non-restrictive large-pore gel
Resolving gel -greater restriction
Have different buffers for stacking gel, resolving gel and tank buffer
What does protein electrophoresis depend on?
The migration of any protein in electricity field depends on pI and pH
What happens to the pI of any given protein?
Constant
What does the pH of a solution determine in protein electrophoresis?
The charge expressed by the protein
What does SDS-PAGE stand for?
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
What type of detergent does SDS-PAGE have?
Strong anionic
Why is the strong anionic detergent important in SDS-PAGE?
- to solubilise, dissociate and denature most proteins to single polypeptide chains
What does SDS-PAGE do to hydrogen bonds?
Disrupts them
What does SDS-PAGE do to hydrophobic interactions?
Blocks them
To what ratio of SDS: protein does SDS-PAGE bind?
1.4:1
What cleaving agents does SDS-PAGE include (with example)?
Disulfide bond (beta-mercapto ethanol)
What is the migration of the protein determined by in SDS-PAGE?
Weight
Where do negatively charged molecules migrate towards in SDS-PAGE?
Positive pole / anode
What determines the effective separation range of SDS-PAGE?
Conc of gel
What is SDS-PAGE not suitable for?
Small polypeptides and peptides with a molecular weight of less than 10kDa
What type of buffer system can be used in protein gel electrophoresis?
Either (continuous or discontinuous)
What are the three protein stains you can use?
Coomassie brilliant blue
Fluorescent stain
silver stain
How does the fluorescent stain work?
Use a fluorescent light to see the samples
What are the ratios of coomassie brilliant Blue needed in electrophoresis?
9:9:2 v/v/v
Methanol, distilled water and acetic acid
How do you compare your sample to known proteins in protein electrophoresis?
You have a band of known proteins running down the side
When is native gel electrophoresis used?
Mainly in circumstances where native conformations are to be analysed (as you don’t have to denature the proteins for it to work)
What is the separation based on in native gel electrophoresis (2)?
Charge: size ratio and conformation (shape)
Give the advantages of native gel electrophoresis (3)
- Potential of separating proteins of identical molecular weight not resolved with SDS-PAGE
- recovery of protein in native state
- study binding events (protein- protein or protein- ligand)
What are the types of nature gels?
Agarose and polyacrylamide
What does SPEP stand for?
Serum protein electrophoresis
What does SPEP measure?
Specific proteins in blood
How does SPEP work?
Uses an electrical field to separate proteins into groups of a similar size, shape and charge
What are the two main groups in blood serum?
Albumin
Globulins
What is a densitometer?
Used to scan separated proteins in the gel- the patterns give info about protein fractions
What is the pH range of haemoglobin electrophoresis?
8-9 (so slightly alkaline)
