Week 12 Flashcards
What is the basic principle of electrophoresis?
Migration of charged particles or molecules in a medium under the influence of an applied electric field
What does the migration rate depend on? (and their effects)
- net charge
- size and shape of particle (smaller moves faster)
- strength of elecrtic field
- properties of medium
- temperature (denaturation, decrease resolution, mixing of analytes, peak broadening)
- ionic strength of buffer
would anything happen if there were no ions in the buffer?
no
what is meant by electrophoretic mobility?
rate of migration (cm/sec) per unit field strength (volts/cm)
what are the roles/properties of the buffer?
- carries applied E field
- set a pH -> preserve our sample, can also influence the migration
- type of charge on solute / extent of ionization of solute
- electrode towards which the solute will migrate
- ionic strength determines the thickness of ionic cloud
3 properties of the supporting medium
- matrix in which separation takes place
- various types exist (paper, gel, …
- separation based on charge and mass of protein depending on the pore size of the medium
give examples of zone electrophoresis and moving boudary electrophoresis
zone:
- paper
- gel
- isoelectric
moving boundary:
- capillary
- isotachophoresis
one advantage and one disadvantage of paper electrophoresis
+: economical, easy to use
-: certain compounds (hydrophilic) can’t be resolved due to the adsorptive and ionogenic properties of paper
gel electrophoresis: what is the molecular sieving based on? one property the supporting medium has to have?
- based on molecular size (gel is porous)
- has to be electrically neutral
agarose gel: what type of molecule, percentage - what does it control, resolution, main usage, main problem
- linear polysaccharide
- 1 to 3 % -> controls pore size (small % = large pores)
- resolution better than paper but less good as polyacrylamide
- nucleic acid electrophoresis
- batch to batch variation is too high
polyacrylamide gel: how it forms, what is needed for it to form, result, main usage, main issue, SDS?
- polymerization of monomer is presence of N,N”-methylenebisacrylamide
- photocatalyzer
- molecular weight in kDa
- protein analysis
- neurotoxic, very dangerous
- it denaturates the protein -> analyse protein mixture qualitatively
what are the two types of gels in a polyacrylamide electrophoresis?
separating gel and stacking gel (where the sample aligns before separating, purpose is to concentrate the protein sample in a sharp band)
4 applications of protein electrophoresis
- measure molecular weight
- peptide mapping
- protein identification / separation
- determine sample purity
what is isoelectrofocusing? and applications?
separation of amphoteric substances (separate proteins which differ in isoelectric point) -> gradient of pH along the length of the gel
- microheterogeneity of proteins
- separate isoenzymes
- in enzymology, immunology
- forensic, food, and agriculture industry
what is a housekeeping gene?
gene that is constantly expressed and we use it as an internal calibrator
what is the main principle of qRT-PCR? what do we visualize?
First reverse transcribe the RNA to cDNA, and then amplify by PCR. We visualize the increase in fluorescence VS the number of cycles.
give the main application of northern, southern and western blotting
northern -> RNA
southern -> DNA
western -> proteins, using labeled antibodies to bind them
