Quantification of protein concentration and chromatography

Question 1

How does the bradford method work and what are its 3 advantages?

Answer 1

the dye forms non-covalent complexes with proteins through electrostatic and Van der Waals interactions with the protein causing a red shift in the
absorbance of the dye from 465nm (red) to 595nm (blue).

Advantages
1. It is sensitive.
2. Very simple: measuring the OD at 595nm after 5 min of
incubation.
3. Vis spectrophotometer can be used.

Question 2

Column Chromatography in general (What are the parameters of separation? What is the stationary and mobile phase? What elutes first?)

Answer 2

Parameters: Molecular Mass, charge and solubility

Sample is passed over a stationary phase (fine granular or bead-like matrix) by the flow of a mobile phase (eluting solvent or eluant)

Those molecules with less interactions to the stationary phase elute first

Question 3

Gel Filtration Chromatography/size exclusion (parameter, what is the stationary phase?)

Answer 3

Separation by molecular mass (size )

Stationary phase: fine beads that contain pores

Question 4

Gel filtration chromatography (how do you calculate Kav, what are the components of Kav?)

Answer 4

Kav = (Ve - Vo)/(Vt-Vo)

Vo is the volume between beads

Ve is the molecules that can enter some of the pores

Vt is the volume of the pores + volume between (for small molecules)

Question 5

What is the purpose of protein quantificatiion (finding out the concentration)?

Answer 5

Determine the progress of the purification of a protein from a mixture of other proteins. Purpose of purification is to increase the specific activity of the enzyme.

Specific activity is the ratio of the enzymatic activity to the total
amount of proteins = units/mg of protein. If a purification is
proceeding properly and the enzyme becomes more pure the
specific activity will increase
▪ Enzymatic activity = units, one unit is the quantity of enzyme
necessary to bring about the formation of 1 µmol of product/min
▪ Total amount of protein, mg

Question 6

Affinity Chromatography (what is it for? what is the stationary phase)

Answer 6

▪ for specific biological molecules: proteins or nucleic acids
▪ The stationary phase contains an immobilized molecule that binds with high specificity to a single or small number of similar proteins
▪ Elution can be done adding high concentration of same molecule immobilized, competing for the binding sites and displacing the purified proteins