physical methods unit 2: electrophoresis

what are some applications for electrophoresis?

• DNA 

• protein 

• IEF

how can we measure DNA with electrophoresis?

• phosphate groups on DNA backbone readily give up protons, nucleic acids are negatively charged, the go from cathode to anode 

• higher voltage increase migration

what are some issues that can arise with DNA electrophoresis?

• if the ionic concentration is too hight is can create too much heat

  • low salt= slower movement but better peaks

• temp of gel

  • effects viscosity of gel

  • increase temp increase diffusion

how does electrophoresis apply to proteins?

• DNA has a lot of charges but proteins are bigger without much charge

  • so we must coat it with charge, SDS PAGE

• proteins move from anode to cathode

what are some issues with electrophoresis and proteins?

• excess O2 can inhibit polymerization elongation

  • we can fix this by deaeration under vaccuum

what are some advantage of electrophoresis for proteins?

small pore size,

what are some instances where protein band doesnt show are expected mw?

• there were some disulfide bonds that werent reduced by mercaptoethanol

• protein was degraded 

• protein has been modified 

• protein has unusual percentage of charged residues 

what is IEF? 

• IEF is another way to measure proteins but by using size and PI

• this is used for proteins that have identical MW but differ in PI or vice versa