Question

1. The following are the properties of severa lhypothetical proteins:

Protein	Charge at pH 9	Molecular weight (Da)
A	-5	40,000
B	-10	40,000
C	-4	160,000
D	-10	160,000

1. a) Predict a pattern of separation of these proteins on a slab native-PAGE.

2. b) Predict a pattern of separation of these proteins on slab SDS-PAGE.

3. c) Predict a pattern of separation of SDS-coated proteins in gel free medium.

Answer 1

In case of native-PAGE, separation of proteins will occur based on charge to mass ratio. Greater the charge to mass ratio greater will be the migration.

But in case SDS-PAGE, separation is based on molecular weight only as proteins are coated with negatively charged SDS , so here intrinsic charge of proteins don't affect the migration.

Separation of SDS coated proteins in gel free medium is also based on molecular weight only.

There are different techniques for protein separation in gel free medium like chromatography, mass spectrometry etc.

Here we have to separate SDS coated proteins in gel free medium so we have to use a technique which separates proteins only based on size / molecular weight. Most widely used method of protein separation depending on molecular weight is size-exclusion chromatography. In size exclusion chromatography , proteins with greater molecular weight follow path of least resistance i.e. a shorter path through the coloumn so come out earlier but proteins with lower molecular weight follow path of greater resistance due to greater diffusion in coloumn so come out later from the coloumn.