Protein electrophoresis Flashcards
What are the ionisible groups in a protein?
• Two ionisable groups; amino gp N-terminus and carboxyl gp C-terminus
Describe charges, what does the charge depend on?
• The charge depends on pH of the buffer.
What is the isoelectric point and what happens when the pH is smaller or higher than this point
• isoelectric point (pI) – The point where the amino acid exists as a zwitterion. The protein has no net charge at this pH, i.e. neutral (zwitterion)
What do R-groups determine?
-the group determines whether amino acid is neutral, acidic or basic
Describe the principles of electrophoresis
• Electrophoresis: - migration of charged particles (macromolecules) in electric field;
migration based on size, shape or charge
current and resistance.
• useful for separating /purifying macromolecules;
• Macromolecules consist of many subunits
each has multiple ion’s
able charged groups.
What are the two types of electrophoresis and what gels do they use?
• Two types:
Horizontal - usually for agarose gel
Vertical - for polyacrylamide gel
What do you need to ensure when choosing apparatus?
- uniform electric field across gel
- cooling to prevent thermal artefact
- access to gel loading and monitoring
What is the purpose of the buffer?
that provides ions to carry current,
maintain relatively constant pH,
pH of solution and nature of R-groups have important effect on migration of proteins,
proteins separated within gel with series of pores.
Describe the types of gels
Agarose
• polysaccharide extract from seaweed.
• prepared by dissolving powdered agarose in buffer.
• heat and pour into casting tray.
• undergoes polymerisation when cooled
• pores relatively larger, so has relatively low resolving power
• can be used to separate large proteins >200kDa
• used at concentrations of 0.5 to 2%
Polyacrylamide
• formed from synthetic small molecule acrylamide
• polymerises into long chains in the presence of catalyst and initiator (APS & TEMED - TEMED – tetra-methyl-ethylene-diamine).
• polyacrylamide gels have smaller pores than agarose.
• pores size also determined by polyacrylamide concentration.
• TEMED – tetra-methyl-ethylene-diamine
What are the two classifications of buffers?
Continuous
Discontinuous
What is a Continuous and Discontinuous gel?
- Continuous buffer system – uses same buffer in gel, sample and tank
- Discontinuous buffer system:- different buffers;
The pI is constant for a protein but what does the pH of the solution determine
The charge expressed by the protein
Describe SDS-PAGE
• SDS-PAGE: most commonly used electrophoretic technique for separation.
• SDS has strong anionic detergent:
to solubilise, dissociate and denature most proteins to single polypeptide chains.
disrupts hydrogen bonds
blocks hydrophobic interactions
• binds at ratio of 1.4g SDS per gram of protein
• conferring net negative charge to polypeptide in proportion to length
• SDS-PAGE includes disulfide bond cleaving agents (e.g. β-mercaptoethanol).
• migration of protein not determined by intrinsic electric charge but by weight
What is the movement of molecules
Cathode to Anode
What determines the separation range?
Gel concentration
Describe native (non-denaturing) electrophoresis
• Used mainly in circumstances where native conformations are to be analysed.
• Native or non-denaturing gels run without SDS.
• Proteins not denatured, separation based on their,
charge-to-size ratio
conformation (shape)
• Charge changes with change in pH of buffer
• Advantages:
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)
• Types of native gels:
Agarose
Polyacrylamide
• Agarose do not have uniform pore size, but optimal for proteins > 200kDa (or nucleic acid >400bp)
• Polyacrylamide gels have uniform pore size, dependent on acrylamide and bis-acrylamide concentrations.
• used for proteins sizes 5 – 2000 kDa
What is the pH range of haemoglobin electrophoresis and what charge will the proteins be?
- pH range 8-9 (slightly alkaline) is most commonly used buffer system;
- majority of proteins will be negatively charged
