campbell7eaise_chapter5

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Paul D. Adams • University of Arkansas

Mary K. Campbell

Shawn O. Farrellinternational.cengage.com/

Chapter FiveProtein Purification and

Characterization Techniques

http://international.cengage.com/

http://international.cengage.com/



Isolation of Proteins from Cells

Many different proteins exists within one cell

• Many steps needed to extract protein of interest, and

separate from many contaminants

• Before purification begins, protein must be released

from cell by homogenization



How We Get Proteins Out of Cells



Salting Out

• After Proteins solubilized, they can be purified based

on solubility (usually dependent on overall charge,ionic strength, polarity

• Ammonium sulfate (NH4SO4) commonly used to “saltout”

• Takes away water by interacting with it, makes proteinless soluble because hydrophobic interactions amongproteins increases

• Different aliquots taken as function of saltconcentration to get closer to desired protein sampleof interest (30, 40, 50, 75% increments)

• One fraction has protein of interest



Differential Centrifugation

• Sample is spun, after

lysis, to separateunbroken cells, nuclei,

other organelles and

particles not soluble in

buffer used

• Different speeds of spin allow for particle

separation



Column Chromatography

• Basis of Chromatography

• Different compounds distribute themselves to a varying

extent between different phases

• Interact/distribute themselves

• In different phases

• 2 phases:

• Stationary: samples interacts with this phase

• Mobile: Flows over the stationary phase and carries

along with it the sample to be separated



Column Chromatography



Size-Exclusion/Gel-Filtration

• Separates molecules based on size.

• Stationary phase composed of cross-linked gel

particles.

• Extent of cross-linking can be controlled to determine

pore size

• Smaller molecules enter the pores and are delayed in

elution time. Larger molecules do not enter and elute

from column before smaller ones.



Size Exclusion/Gel-filtration (Cont’d)



Affinity Chromatography

•Uses specific binding properties of molecules/proteins

•Stationary phase has a polymer that can be covalently

linked to a compound called a ligand that specifically

binds to protein



Group Specific Affinity Resins



Ion Exchange

• Interaction based on overall charge

(less specific than affinity)

• Cation exchange

• Anion exchange



Electrophoresis

• Electrophoresis- charged particles migrate in electric

field toward opposite charge

• Proteins have different mobility:

• Charge

• Size

• Shape

• Agarose used as matrix for nucleic acids

• Polyacrylamide used mostly for proteins



Electrophoresis (Cont’d)

• Polyacrylamide has more resistance towards larger

molecules than smaller

• Protein is treated with detergent (SDS) sodium

dodecyl sulfate

• Smaller proteins move through faster (charge andshape usually similar)



Isoelectric Focusing

• Isolectric focusing- based on differing isoelectric pts.

(pI) of proteins

• Gel is prepared with pH gradient that parallels electric-

field. What does this do?

• Charge on the protein changes as it migrates.

• When it gets to pI, has no charge and stops



Primary Structure Determination

How is 1˚ structure determined?

1) Determine which amino acids are present (amino

acid analysis)

2) Determine the N- and C- termini of the sequence

(a.a sequencing)

3) Determine the sequence of smaller peptide

fragments (most proteins > 100 a.a)

4) Some type of cleavage into smaller units necessary



Primary Structure Determination



Protein Cleavage

Protein cleaved at specific sites by:

1) Enzymes- Trypsin, Chymotrypsin

2) Chemical reagents- Cyanogen bromide

Enzymes:

Trypsin- Cleaves @ C-terminal of (+) charged side

chains

Chymotrypsin- Cleaves @ C-terminal of aromatics



Peptide Digestion



Cleavage by CnBr

Cleaves @ C-terminal of INTERNAL methionines



Determining Protein Sequence

After cleavage, mixture of peptide fragments produced.

• Can be separated by HPLC or other chromatographic

techniques

• Use different cleavage reagents to help in 1˚ determination



Peptide Sequencing

• Can be accomplished by Edman Degradation

• Relatively short sequences (30-40 amino acids) can

be determined quickly

• So efficient, today N-/C-terminal residues usually not

done by enzymatic/chemical cleavage



Peptide Sequencing

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