LEC 8: Protein Analysis Flashcards
What are the 3 general steps for purifying a protein
- Disrupt cells and obtain an enriched protein fraction
- Purification
- Monitoring the progress and success of the purification scheme
Step 1: Cell lysis
What it’s characteristics (3)
- Grinding (mortal and pestle)
- Sonication (high frequency sound)
- Detergents to disrupt membranes
Step 1: Cell lysis
Define the differential centrifugation
Extraction of the protein pool
Step 2: Purificaiton
What are the 3 Column chromatography
- Size-exclusion (gel- filtration)
- Ion-exchange
- Affinity
Principles of column chromatography
A protein mixture is carried in a ___ solution through a ___ column of ___
Buffer
Porous
Small beads (matrix)
Principles of column chromatography
Protein are separated as a result of ___ with the column matrix
Differential interactions
Step 2: Purificaiton
What are the characteristics of Size-exclusion (Gel filtration) chromatography (4)
- Separates proteins on the basis of their size
- Column made of porous beads
- Small beads can enter
- Large proteins travel faster through column
Step 2: Purificaiton
What are the characteristics of Ion-exchange chromatography (2)
- Separates proteins on the basis of their change
2. Net charge at pH 7
Proteins with ___ will have a net (+) charge at pH 7 leads to ____ proteins
pI>7
Basic
Proteins with ___ will have a net (-) charge at pH 7 ->____ proteins
pI<7
Acidic proteins
Step 2: Purificaiton
What are the characteristics of Affinity chromatography
- Separates proteins on the basis of their ability to bind other molecules
Protein that covalently bind other molecules are called___
Ligands
Step 3: Checking the efficiency of the purification scheme
What are the characteristics of Gel electrophoresis
- Helps identify the quantity and approximate mass of proteins in a sample
What does SDS-Page do
Separates proteins based on their mass only
