Analytical techniques

Question 1

Convert Nitrogen content to protein content, what it is used?

Answer 1

The nitrogen to protein conversion factor N-prot factor, g of protein/ g od nitrogen, but often a value of 6.25 is used.

Question 2

Molecular weight distribution:

Answer 2

1. SDS-PAGE, for protein composition: which and how much of each, individual proteins are present in the sample. SDS surfactant that causes folded proteins to unfold and gives all the proteins a simlilar charge-to-mass ratio. Only individual polypetide chains are shown in the gel. Electric field, larger proteins move more slowely. E.g : caseins higher Mw thatn lactoglobulins.
2. Size- exclusion chromatography: injected into a column. Will be separated on their hydrodynamic volume ( related to the molecular weight). `NATIVE´ conditions, no surfactants or reducing agents only buffer. Any aggregates or complex NOT dissociated.

Question 3

Measure solubility:

Answer 3

1. Measured and disolve under solubility.
2. pH adjusted with buffers.
3. Centrifuged (to precipitate proteins that became insoluble).
4. Determine the concentration od the supernatant with a colorimetric assay (Biuret).

Question 4

Free amino group content- Degree of hydrolysis.

Answer 4

Hydrolysis of the petide bonds–> liberation of free amino acids (colorimetric assays , OPA) and free carboxylic groups.