W10 Electrophoresis Flashcards
isoelectric point (pI)
protein has no net charge at this pH, i.e. neutral (zwitterion)
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 ionisable charged groups
Electrophoresis apparatus
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
Gel Electrophoresis
Gel usually cast in shape of thin slab with wells,
immersed within 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
Agarose
Electrophoretic gel
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).
polyacrylamide gels have smaller pores than agarose.
pores size also determined by polyacrylamide concentration
Vertical slab gel electrophoretic apparatus
Most popular technique for protein electrophoresis
Buffer
supplies current carrying ions in electrophoretic cell
maintains desired pH
provides medium for heat dissipation
Buffer systems classified as
Continuous
Discontinuous
Continuous buffer system
Uses same buffer in gel, sample and tank
Discontinuous buffer system
Non-restrictive large-pore gel
Resolving gel – greater resolution
Have different buffers for stacking gel, resolving gel and tank buffer
Protein Electrophoresis
Migration of any protein in electric field depends pI and pH
pI is constant for any given protein
pH of solution determines the charge expressed by protein;
e.g. Hb with pI of 7.07 donates proton to buffer if placed in electrophoretic buffer of pH 8.6
SDS-PAGE
most commonly used electrophoretic technique for separation
SDS-PAGE includes disulfide bond cleaving agents (e.g. β-mercaptoethanol).
migration of protein not determined by intrinsic electric charge but by weight
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
Choice of Gel concentration
Gel concentration determines effective separation range of SDS-PAGE
SDS-PAGE not suitable for small polypeptide and peptides, of MW <10kDa
Continuous or discontinuous buffer system can be used in protein gel electrophoresis
Detection Methods
Protein staining (in situ) Coomassie brilliant blue dyes used as 0.1% (w/v) in methanol, distilled water and acetic acid (9:9:2,v/v/v)
Native (non-denaturing) Gel 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
Native (non-denaturing) Gel electrophoresis - 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 pore size
do not have uniform pore size, but optimal for proteins > 200kDa (or nucleic acid >400bp)
Polyacrylamide pore size
Polyacrylamide gels have uniform pore size, dependent on acrylamide and bis-acrylamide concentrations.
used for proteins sizes 5 – 2000 kDa
Serum Protein Electrophoresis (SPEP) background
Blood made up of blood cells and plasma
Plasma is made up of water, proteins, salts, glucose, hormones, clotting factors
Serum Protein Electrophoresis (SPEP) test
This measures specific proteins in blood;
SPEP uses electrical field to separate proteins into groups of similar size, shape and charge;
It helps to identify diseases
Blood serum contains 2 major protein groups
Albumin
Globulins
Densitometer
Used to scan separated proteins in the gel. The pattern gives information about protein fractions
Haemoglobin Electrophoresis
pH range 8-9 (slightly alkaline) is most commonly used buffer system;
majority of proteins will be negatively charged
