Biorad Protein Analysis - allows separation of proteins in active state and can

resolve proteins in same MW

Electrophoresis - preserves native charge-to-mass ration, subunit
- cm2/ Vsec: depends on the electrophoresis system and proteins interactions, and biological activity
- factors: electric field, temperature of the system, pH, ion type, - protein preparation and electrophoresis are performed in:
buffer concentration, size-shape-charge of proteins non-reducing, nondenaturing sample buffer
- applications
 purification - has unpredictable separation because:
 assessing protein purity  separate multisubunit complexes
 gathering data on protein regulation  migration towards any pole of the electrode
 protein size determination - limitations:
 isoelectric point  not for MW determination
 enzymatic activity  SDS-PAGE
- once proteins are separated, downstream applications: - Leammli incorporated Sodium Dodecyl Sulfate (SDS) in
 enzymatic assays the discontinuous buffer system
 more purification - SDS is a lipid molecule that has a charged head, acting as
 transfer to membrane for immunological detection: a surfactant
western blot and immunoblotting - SDS is only required in protein preparation sample buffer
 elution and digestion: mass spectroscopy (not in the gel buffer); and it does the following:
- Workflow:  shape: denatures the protein, making it rod-
1. Method Selection shaped and linearized: removing the tertiary
2. Sample Preparation conformation
3. Gel and Buffer Preparation  charge: gives the protein an overall negative
4. Performing Electrophoresis charge so it only goes to the anode (+)
5. Protein Detection and Analysis  same charge to mass ratio: 1.4 g SDS binds to 1h
protein, making a constant 1 SDS: 2aa
Agarose Gel Electrophoresis (AGE) Isoelectric Focusing
- for large proteins or protein complexes >300 kD - electric field + pH gradient: separation according to isoelectric
point (pI)
- highest resolution to all electrophoresis techniques
- when the protein encounters
 pH gradient: net charge changes
 electric field: migrates to where its net charge is zero
Native IEF: retains protein structure and enzymatic activity
Denaturing IEF: presence of high concentrations of urea; high
resolution and more suitable for complex protein analysis
 Urea dissociates proteins into individual subunits and
abolishes secondary and tertiary structures

PAA: positive, anode, acidic

BeN Cab: basic, negative, cathode

Polyacrylamide Gel Electrophoresis (PAGE)

- smaller proteins travel more rapidly
- for protein size range of 5-250 kD
- vertical orientation
Types of Buffer Systems:
 Continuous Buffer System
- same buffer in the gel, sample, and electrode reservoirs
- used for nucleic acid analysis
 Discontinuous Buffer System
- stacking gel: large pores
- resolving gel: small pores
- different buffers in gels and electrode solutions
- has higher resolution than continuous bec:
 proteins migrate quickly in stacking gel
 slowed down and becomes a thick band upon
meeting the resolving gel Two Methods to generate stable, continuous pH gradient
 has ions that sandwich the proteins 1. Carrier Ampholytes: heterogenous mixtures of small conductive
 Discontinuous Native Page polyamino-polycarboxylate compounds that carry multiple charges
- developed by Ornstein and Davis with closely spaced pI values. Voltage causes alignment of
ampholytes to their PIs causing pH gradient
 Ampholytes: molecule containing both acid and base  Zwitterionic detergents: CHAPS, sulfobetaines (for integral
properties membrane proteins)
 Anionic detergent: SDS (used for PAGE, best solubilization for
2. Immobilized pH Gradients (IPG) strips: formed by covalently proteins)
grafting buffering groups to a polyacrylamide gel backbone to
generate a stable pH for different pH ranges and gradients. Reducing Agents: disrupt intra- and intermolecular disulfide bonds
to achieve complete protein unfolding
 made with buffering acrylamide derivatives that contain  2-mercaptoethanol (BME): volatile and used in large excess to
either a free carboxylic acid or a tertiary amino group prevent reoxidation
 copolymerized with acrylamide and bis-acrylamide  dithiothreitol (DTT): less volatile favors protein reduction and
 pH gradient is precast into the gel and cannot shift during used in small amounts
electrophoresis.  Phosphines (trubutylphosphine, tris-carboxyethylphosphine)
- thiol reducing agents and can be used at lower
2D Electrophoresis concentrations
 allows separation of thousands of polypeptides in a single
slab gel Chaotropic Agents
1. Separation based on pI  Urea (and Thiourea): disrupt H-bonds and hydrophobic
 uses denaturing IEF or IPG gel strip interactions destroying secondary structure
2. Separation based on Weight - can be used in SDS-PAGE
 uses SDS-PAGE - protein solution should not be heated above 37 deg C
because urea and thiourea get hydrolyzed and can modify
Preparative Electrophoresis amino acids (carbamylation) giving rise to. artifactual charge
- separate large amounts of protein for the purposes of purification heterogeneity
or fractionation
- can be done via PAGE or 2D Electrophoresis Buffer and Salts
 Tris Base: used since most proteins are soluble at higher pH
Sample Preparation for Electrophoresis
3. Removal of Interfering Substances: salts, detergents,
General Conditions denaturants, or organic solvents, high DNA and carbohydrate
- simplify workflow to prevent variability content (viscous)
- include protease inhibitors  Protein Precipitation: protein precipitation with
- solubilize in a buffer corresponding to electrophoresis technique trichloroacetic acid / acetone
- use protein extracts immediately or store in -80 deg C freezer to  Buffer Exchange: size exclusion chromatography removes
avoid freeze-thaw cycles salts, detergents, and other contaminants

1. Cell Disruption
- disruption of cells with tough cell walls include harsher methods: 4. Sample Quantitation (Protein Assays): determines the
sonication, French press, grinding, mechanical homogenization, concentration of the protein to ensure appropriateness for
glass-bead homogenization electrophoresis and visualization; uses colorimetric assays
- causes release of hydrolases, to avoid this: - includes Bradford, Lowry, and BCA
 disrupt cells in strong denaturing reagents: Urea, SDS,
Thiourea 5. Preparation for PAGE
 perform disruption at low temperatures or basic pH:
enzymes are least active Reagent Selection and Preparation
 add cocktail of protease inhibitors: EDTA, benzamidine,
phenylmethylsulfonyl fluoride (PMSF), aminoethyl- Polyacrylamide Gels
benzene sulfonyl (AEBSF), tosyl lysine chloromethyl ketone - stable, chemically inert, electrically neutral, hydrophilic, and
(TLCK), tosyl phenyl chloromethyl etone (TPCK) transparent for optical detection at wavelengths greater than
 for protein phosphorylation studies: fluoride and vanadate 250nm.
- then, check efficacy of cell wall disruption and centrifruge extracts - does not interact with solutes and has low affinity for common
to remove insoluble materials, which can block the gel pores protein stains

2. Protein Solubilization
- proteins interactions should be broken down to prevent Polymerization of Polyacrylamide Gel
aggregation resulting in artifacts or sample loss
- interactions such as disulfide bonds, ionic interactions, H-bonds,
- free-radical copolymerization of the mixture of
Van der Waals etc.
- use appropriate buffer and add detergents, chaotropic agents, acrylamide and a cross-linker bis-acrylamide
reducing agents, salts, and ampholytes
- catalyst initiator system, ammonium persulfate (APS)
Detergents and tetramethylethylenediamine (TEMED)
- disrupts hydrophobic interactions between and within proteins
 Nonionic detergents: NP-40, Triton X-100 (not for hydrophobic
proteins)
- TEMED sped up the rate of formation of free radicals
from ammonium persulfate.
- persulfate free radicals converted monomers of
acrylamide to another free radical which reacted with
inactivated monomers, initiating the polymerization
reaction.

-
The elongating polymers were then randomly
crosslinked by bis, resulting in a gel with a characteristic
porosity that was dependent on the relative concentration
of acrylamide to cross-linker and in the polymerization
conditions

Figure 1. Polyacrylamide gel polymerization reaction.

The double-bonded structures of acrylamide and bis-

acrylamide are appropriate for radical polymerization
reactions. The radical formed upon the attack of a
terminal carbon also reacted with another acrylamide
monomer at the terminal position. The results are the
formation of long fibers of extended polyacrylamide
called “gel.” Polymerization propagates indefinitely until
terminated by reaction with molecular oxygen, another
radical end of a polyacrylamide chain, or the walls of the
container.