Biochemistry Ch 1

Methods for determining protein presence/ purity

1. SDS-PAGE

2. Protein function Assay

3. Immunological methods (elisa or western blot)

4. Mass spec

SDS page or polyacrylamide gel electrophoresis with protein stain

pros- fast inexpensive can see al proteins in sample

cons- need a decent amount of protein. cant tell if protein is folded/ active

Methods for determining protein presence/ purity

Protein Function Assay (ie oxygen binding if the protein is hemoglobin)

pros- tells which fraction contain active and folded form of your protein

cons- you need to compare the activity to the total protein concentration to determine purity. doesnt give you any information on what the impurities are.

Methods for determining protein presence/ purity

Immunological methods (elisa or western blot)

Pros- more sensitive than PAGE so it can detect smaller amounts of your protein

Cons- only detects your protein that you blot for so you cant tell if it is pure

Methods for determining protein presence/ purity

Mass Spectrometry

Pros-give you more exact mass of your protein and contaminants

Cons- amounts are not quantitative! (can’t tell if contaminants are 10% or 50% of total protein concentration)

Methods for determining protein presence/ purity

Chromatography types

1. cation exchange

2. anion exchange

3. reverse phase/ hydrophobic exchange

4. gel filtration

Cation exchange

Separates based on: charge

Buffer pH: pH<pI

Loading buffer: low salt

Elution buffer: high salt

Anion Exchange:

Separates based on: charge

Buffer pH: pH>pI

Loading buffer: low salt

Elution buffer: High salt

Reverse Phase/ Hydrophobic Exchange

Separates based on: hydrophobicity

Buffer pH: pH=pI

Loading buffer: High Salt

Elution buffer: Low salt

Gel filtration/ size exchange

Separates based on: size

Buffer pH: pH > or < pI

Loading buffer: some salt prevent non specific binding

Elution buffer: same as loading

C term pka

2

N term pka

9

Asp and Glu pka

4

His

6

cys pka

8

lys, tyr pka

10

arg pka

12

Bohr Effect

The Bohr effect describes hemoglobin's reduced affinity for oxygen in response to a lower blood pH and increased carbon dioxide levels

Competitive Inhibitor

• Binds: Active site

• Km: ↑ (decreases affinity)

• Vmax: ↔ (no change)

Non Competitive inhibitor

• Binds: Allosteric site (enzyme ± substrate)

• Km: ↔ (no change)

• Vmax: ↓

Un competitive inhibitor

• Binds: Only ES complex (allosteric site)

• Km: ↓

• Vmax: ↓