Why would you need to purify protein?

Methods for Working with Protein

1. Protein IsolationA. Selection of a protein source

i. tissue and cell cultures (bacteria, yeast, mammalian, etc.)ii. genetically engineered - tagged proteins, over-expression

B. Solubilizationi. osmotic lysis - hypotonic solution

swelling and burstingii. French press - high pressure & small orificeiii. sonicatoriv. homogenizer -tissue grinder, v. glass beads versus mortar & pestlevi. dounce - .

What properties of proteins can be used to separate and purify

them from each other?

Methods for Working with Protein

2. Separation methodsA. Properties that are used to separate proteins:

i. chargeii. hydrophobicityiii. affinityiv. solubility & stabilityv. molecular weight

B. differential centrifugation - S-100 versus S-30C. precipitation/solubility

i. salting in versus salting outsolubility 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 solutionS` - 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 methodsD. chromatography - overall example

i. ion exchange - cation vs. aniona. strong versus weak (effect of pH) b. counterion present is importantc. types of gradients and their application

linear, nonlinear, step

Methods for Working with Protein

2. Separation methodsD. chromatography - overall example

ii. affinity chromatographya. ligand based:

glutathione covalent linked to resinGST fusion protein

b. speciality dyes - Cibracon blue and othersc. Immunoaffinity:

epitope tags such as FLAG, V5, etc.d. DNA - general and specific DNAse. others (example: heparin, hydroxyapatite)

Methods for Working with Protein

2. Separation methodsD. chromatography

iii. gel exclusion or gel filtrationa. separation based on sizeb. exclusion volumec. different size limit materials

iv. HIC or hydrophobic interaction chromatographycompare to reverse phase chromatography

v. types of resin cellulose, dextran, agarose, polyacrylamide, perfusion

beads

Sequence Databases

Some specifically for proteins and others for DNA

http://www.ncbi.nlm.nih.gov/sites/entrez?db=PubMed&itool=toolbar

http://blast.ncbi.nlm.nih.gov/Blast.cgi

PubMed – access to multiple databases

BLAST search

Gel filtration or size exclusion chromatography

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 problems?

Methods for Working with Protein

3. Stabilization of proteinA. Changing buffer

i. dialysis ii. ultracentrifugation

B. Concentrating proteinhelps to maintain active proteinsometimes addition of a carrier protein may helpaddition of glycerol

C. Inhibitorsi. proteasesii. phosphatase inhibitors

Methods for Working with Protein

4. Protein AnalysisA. Gel electrophoresis

i. Discontinous gel a. polyacrylamide:

ratio bisacrylamide/ acrylamideb. TEMED - free radical stablizerc. ammonium persulfated. stacking gel pH 6.8, contains glycine pK2=9.78e. running gel is pH 8.8, sample contains

bromophenol blue

Methods for Working with Protein

4. Protein AnalysisA. Gel electrophoresis

ii. SDS-PAGEa. SDS forms a micelle around the polypeptide

the size and charge of the micelle is approximately proportional to the size of the polypeptide

b. denatures proteinsc. 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, glycinemoves 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 AnalysisB. 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 AnalysisC. ImmunoblottingD. Gel-shift or EMSA

(electrophoretic mobility gel shift assay) assays

Western blotting