Electrophoresis Flashcards
Describe how proteins can have a charge.
- Proteins can have either a net positive or negative charge i.e due to ionisable amine and carboxyl groups
- Charge depends on the pH of the buffer.
- In acidic solution, it has a net positive charge.
- In basic solution, it has a net negative charge
- Backbone of the protein is uncharged. R group determines whether an amino acid is neutral, acidic or basic.
Based on the isoelectric point (pI)
- the pH at which the protein will have no net charge i.e found as a zwitterion
- constant - does not change
- if pH < pI , protein has net positive charge
- if pH > pI, protein has net negative charge
What is electrophoresis?
- Migration of charged molecules (macromolecules) in an electric field so that they can be separated or purified - determined by pH and pI
- Macromolecules are made up of subunits with multiple ionisable groups e.g polypeptides made up of amino acids joined by peptide bonds
- Migration based on size, shape or charge
- Vertical slab gel electrophoresis - current most popular technique
Describe the two types of electrophoretic gel.
AGAROSE (HORIZONTAL)
- Polysaccharide extract from seaweed.
- Prepared by dissolving powdered agarose in a buffer, heating then pouring into a casting tray.
- Undergoes polymerisation when cooled.
- Pores are relatively larger, so it has a relatively low resolving power.
- Can be used to separate large proteins of over 200kDA
- Used at concentrations of 0.5-2%.
POLYACRYLAMIDE (VERTICAL)
- Formed from the synthetic small molecule acrylamide.
- Polymerises into long chains in the presence of a catalyst and initiator (APS and TEMED(tetra methyl ethylene diamine)
- Polyacrylamide gels have smaller pores than agarose.
- Pore size is also determined by the polyacrylamide concentration.
Why are buffers used during electrophoresis?
A buffer:
- supplies current carrying ions in electrophoretic cells
- maintains the desired pH
- provides a medium for heat dissipation
How can buffers be classified?
CONTINUOUS: uses the same buffer in the gel, sample and tank
DISCONTINUOUS: uses different buffers for stacking gel, resolving gel and the tank buffer
- Non-restrictive large-pore gel
- Resolving gel -greater restriction
Describe SDS-PAGE.
- Stands for sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
- Most commonly used electrophoretic technique for separation.
- Uses disulfide bond cleaving agents e.g β-mercaptoethanol
SDS has strong anionic detergent:
- to solubilise, dissociate and denature most proteins to single polypeptide chains
- to disrupt hydrogen bonds
- to block hydrophobic interactions
Migration of the protein is not determined by electrical charge, but by weight/molecular size
- Negatively charged molecules will migrate towards the anode
- Effective separation range determined by gel concentration
- Binds at ratio of 1.4g SDS per gram of protein
SDS-PAGE isn’t suitable for small polypeptides and peptides with a molecular weight of less than 10 kDa
Either a continuous or discontinuous buffer system can be used in SDS-PAGE and protein gel electrophoresis
Describe native gel electrophoresis.
- Used mainly in circumstances where native conformations are to be analyzed and biological activity needs to be preserved. These native/non-denaturing gels run without SDS.
Proteins aren’t denatured. Separation is based on their:
- charge-to-size ratio
- conformation (shape)
The advantages are:
- the potential of separating proteins of identical molecular weight, not done with SDS-PAGE
- recovery of the protein in the native state
- able to study binding events (eg. protein-protein or protein-ligand)
Both agarose and polyacrylamide gels can be used in this technique. They are used for different sized molecules: agarose for proteins >200 kDa and don’t have uniform pore size, polyacrylamide for 5-2000 kDa and have uniform pore sizes (dependent on acrylamide concentration)
Give an example of the clinical applications of electrophoresis.
SERUM PROTEIN ELECTROPHORESIS (SPEP)
- Blood is made up of blood cells and plasma. Plasma is made up of water, PROTEINS, salts, glucose, hormones, clotting factors, etc.
- Measures specific proteins in the blood. SPEP uses an electrical field to separate proteins into groups of similar size, shape and charge.
- Helps to identify diseases. Blood serum contains two major protein groups: Albumin and Globulins.
A densitometer is used to scan separated proteins in the gel. The pattern gives information about protein fractions.
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
Outline what occurs in gel electrophoresis
- Gel usually cast in shape of thin slab with wells, immersed within buffer:
- Buffer provides ions to carry current, to maintain relatively constant pH,
- pH of solution and nature of R-groups have important effect on charge expressed by proteins and therefore, extent of migration of proteins,
- Proteins separated within gel with series of pores
How can proteins be detected during electrophoresis?
- Protein staining (in situ) e.g fluorescent staining - use of fluorescent lighting to view samples
- Band of known proteins running down the side to compare sample to known proteins
- Coomassie brilliant blue dyes used as 0.1% (w/v) in methanol, distilled water and acetic acid (9:9:2,v/v/v)
What is the pH range used in Hb electrophoresis?
Suggest what the effect would be on the proteins involved.
- pH range 8-9 (slightly alkaline) is most commonly used buffer
- Majority of proteins will be negatively charged
Outline the function of the following during electrophoresis.
- Tank
- Power block
- Sample comb
- Casting tray
Tank
- Where the sample and buffer are attached to a power block
Power block
- Supplies electric current through the buffer
Casting tray
- Preparing the gel
Sample comb
- Makes an indentation in the gel that allows you to put your sample in the buffer before applying the current
