Methods for Working with Proteins

Why would you need to purify
Methods for Working with Protein
1. Protein Isolation
A. Selection of a protein source
i. tissue and cell cultures (bacteria, yeast, mammalian, etc.)
ii. genetically engineered - tagged proteins, over-expression
B. Solubilization
i. osmotic lysis - hypotonic solution
swelling and bursting
ii. French press - high pressure & small orifice
iii. sonicator
iv. homogenizer -tissue grinder,
v. glass beads versus mortar & pestle
vi. dounce - .
What properties of proteins can
be used to separate and purify
them from each other?
Methods for Working with Protein
2. Separation methods
A. Properties that are used to separate proteins:
i. charge
ii. hydrophobicity
iii. affinity
iv. solubility & stability
v. molecular weight
B. differential centrifugation - S-100 versus S-30
C. precipitation/solubility
i. salting in versus salting out
solubility of a protein close to its pI versus
the effect of salt interacting w/ solvent & not the protein
ii. examples of
a. ammonium sulfate precipitation
b. PEI - poly(ethyleneimine)precipitation
S – solubility of protein in salt solution
S` - solubility of protein in pure water
Protein Solubility
• Salt-in
– At lower ionic strength, increased salt
increase solubility
• Salt-Out
– At higher ionic strengths
– Increased salt concentrations causes the
protein to precipitate out of solution
– Competition between the added salt with
other dissolved solutes for solvation
Solute-solute interactions > solute-solvent interactions
Solubility of lactoglobulin depends on pH
Methods for Working with Protein
2. Separation methods
D. chromatography - overall example
i. ion exchange - cation vs. anion
a. strong versus weak (effect of pH)
b. counterion present is important
c. types of gradients and their
linear, nonlinear, step
Methods for Working with Protein
2. Separation methods
D. chromatography - overall example
ii. affinity chromatography
a. ligand based:
glutathione covalent linked to resin
GST fusion protein
b. speciality dyes - Cibracon blue and others
c. Immunoaffinity:
epitope tags such as FLAG, V5, etc.
d. DNA - general and specific DNAs
e. others (example: heparin, hydroxyapatite)
Methods for Working with Protein
2. Separation methods
D. chromatography
iii. gel exclusion or gel filtration
a. separation based on size
b. exclusion volume
c. different size limit materials
iv. HIC or hydrophobic interaction chromatography
compare to reverse phase chromatography
v. types of resin
cellulose, dextran, agarose, polyacrylamide, perfusion
Sequence Databases
Some specifically for proteins and
others for DNA
PubMed – access to
multiple databases
BLAST search
Gel filtration or size exclusion
What is the size of the protein of
interest and the contaminating protein?
How best to separate them?
Precautions or concerns might
you have about maintaining your
protein’s activity
What can you do to avoid these
Methods for Working with Protein
3. Stabilization of protein
A. Changing buffer
i. dialysis
ii. ultracentrifugation
B. Concentrating protein
helps to maintain active protein
sometimes addition of a carrier protein may help
addition of glycerol
C. Inhibitors
i. proteases
ii. phosphatase inhibitors
Methods for Working with Protein
4. Protein Analysis
A. Gel electrophoresis
i. Discontinous gel
a. polyacrylamide:
ratio bisacrylamide/ acrylamide
b. TEMED - free radical stablizer
c. ammonium persulfate
d. stacking gel pH 6.8, contains glycine pK2=9.78
e. running gel is pH 8.8, sample contains
bromophenol blue
Methods for Working with Protein
4. Protein Analysis
A. Gel electrophoresis
a. SDS forms a micelle around the polypeptide
the size and charge of the micelle is approximately
to the size of the polypeptide
b. denatures proteins
c. protein stains: Coomassie blue, silver stains,
fluorescent stains
d. gradient gels
SDS-PAGE –principle
•Glycine is an amino acid, and its charge
property changes depend on the pH.
•In stacking gel (pH6.8), only a little amount
of glycine is negatively charged, thus, glycine
moves very slowly.
•SDS-bound proteins move much faster in
low-density gel. Thus, proteins are stacked
on the running front of glycine.
•In separation gel (pH8.8), glycine is charged
and moves very fast. Proteins can move
dependent on their size.
•Stacking gel 5%, pH6.8
•Separation gel 6-20%, pH8.8-+
Methods for Working with Protein
4. Protein Analysis
B. 2-Dimensional gels
1st dimension: isoelectric focusing
tube gel format
ampholytes to create an immobilized pH gradient
2nd dimension is usually SDS-PAGE
General Formula of Ampholytes
•Each ampholyte has a different pK and isoelectric point.
•Each ampholyte has a particular buffering capacity
Methods for Working with Protein
4. Protein Analysis
C. Immunoblotting
D. Gel-shift or EMSA
(electrophoretic mobility gel shift assay) assays
Western blotting