lecture 7 - purification with isoeclectric focusing

Question 1

how does isoelectric focusing (IEF) work?

Answer 1

• tube containing an electric feild is embeded with a fixed pH gradient
• proteins migrate to the location where their pH = pI

Question 2

IEF is typically combined with what? to form what?

Answer 2

SDS-PAGE to form 2-D electrophoresis

Question 3

how do we interpret the SDS-PAGE after IEF? (horizontal vs vertical)

Answer 3

• proteins in the same horizontal range have similar size but different pI
• proteins in the same vertical range have similar pI but different sizes

Question 4

how do multimers migrate on IEF? on SDS-PAGE?

Answer 4

• migrate on IEF according to the pI of the full protein

- migrate on SDS-PAGE according to mass of individual subunits

Question 5

why is 2D electrophoresis useful?

Answer 5

• can separate 1000s of proteins

- often combined with mass spectophotometry and used for proteomics