08 Protein Purification and Analysis Pt. 2 Protein Analysis

protein analysis

-determination of protein concentration
-Determination of number of subunits
-molecular weight fetermination
-assessment of biological activity

biuret assay

Reagent; strongly alkaline solution containing Cu2+
Forms: purple colored soln upon complex formation of Cu2+ with at least 3 amino acids residues
Purple complex absorbs at 540nm

Directly

Intensity of purple volor is _____ proportional to the amount of proteun

Bradford assay

Reagent: dye coomasie brilliant blu G-250
Dye binding method
Binda thru hydrophobic and electrostatic interaction
Form, initial color red
But upon reaction it turns blue
Blue color absorbs at 595 nm

Lowry Assay modified biuret assay

Reagent: biuret plus folin-ciocalteau Reagent
Formation of biuret and reduction of folin
Initial color yellow
But changes to blue

detected at 750nm

Commonly used standard protein solution

Bovine serum albumin

Gel Electrophoresis

separate proteins based on net electric charges and molecular weight
Assess the purity
Determines number of subunits
Involves migrating of proteins on a porous matrix upon application of electric field

Commmonly used for proteins

Polyacrylamide gel electrophoresis (PAGE)

gel electrophoresis

Absence of debaturaing agent (Native PAGE), use to asses purity
Prwsnec of denaturaing agent (SDS-PAGE), use to determine number of subunits

Gel electrophoresis

Smallest molecules migrates the fastest shile larger molecules migrates sliwer beacsuse entangles with porour matrix

Native PAGE

Assessment of purity
Purity we must only see one band if there are any other band pa edi hindi pa purified
Migration of proteins are dictated by cahrge and molecular weight

SDS-PAGE

Sodium dodecyl sulfate PAGE
determines number and molecular weight of subunits
Used when there is one band from native page

SDS (anionic detergent)
Beta-mercaotoethanol or dithiothreitol (for reduction of disulfide bind)
Migration is dictated by molecular weight only

SDS is done in the presence of following denaturants

Salt concentration (ionic strength)

Solubility of proteins is strongly dependent on the ________ of the medium.

Poorly

Proteins are usually _______ soluble in purewater

Increases, aggregation

Protein solubility increases as the ionic strength _______, because more and more of the well-hydrated inorganic ions are bound to the protein's surface, preventing _______ of the molecules (salting in).

Small, larger

Dialysis, the main feature of this membrane is that it is porous, with thee pore size that _____ salt ions can freely pass, while ____ proteins cannot