Techniques in Protein
Biochemistry
Chapter 5
• Problem: isolation & analysis of protein or amino
acid found in cell
• Assumption: can somehow analyze for wanted
protein
– Common – Colorimetric indicator (chemical rxn 
color form’n; can be monitored spectrophotometrically)
– Functional indicator (biological endpoint)
– This example – colorimetric (breakdown of fats 
purple color)
• Activity assay
• Use at each step of separation
Isolation of Wanted Protein from
Brain Cells
• Brain cells contain wanted
protein
• Open cells
– Homogenization,
sonication, grinding
– Maintain cold, pH,
osmolality
• Centrifugation often used
Known speeds/
conditions for
different organelles
• Test each fraction for activity
– Save most active fractions
• Separation of wanted protein from other
types of molecules
– Dialysis against physiological buffer
Separation from other proteins
A. Chromatography
All use solid or aqueous support to which
wanted protein has some affinity
All use aqueous or gaseous mobile phase;
wanted protein has different affinity
This also moves molecules through/ past
support
• If wanted protein has greater affinity for
support than for mobile phase, protein
“adheres” to support phase
• If wanted protein has greater affinity for
mobile phase than for support, protein will
move with mobile phase through/away from
support
Gel Filtration Chromatography
(= Size Exclusion)
• Separation by MW
• Solid support = porous beads (ex: sephadex,
sepharose)
– Held in column
– Beads have microscopic pores/pits/spaces
• Mobile phase = buffer of physio pH, ionic
strength
• Sample = sol’n of wanted protein + other
(unwanted) proteins; most have different
MWs
• Apply sample to column
• Begin slow mobile phase flow
– Smaller proteins enter spaces in beads
– Larger proteins flow w/ buffer around beads (so
emerge 1st from column)
• Collect fractions; test each fraction by
activity assay
Ion Exchange Chromatography
• Separation by overall charge of proteins
• Solid support = resin (charged microscopic beads)
suspended in buffer
• Mobile phase = buffer of particular pH, ionic
strength
• Sample = sol’n of wanted protein + other
(unwanted) proteins
Most have different overall + or – charges of
various strengths
• Apply sample to column, begin slow mobile
phase flow
– Proteins of charge opposite that of resin + of
similar strength of charge of resin: Good
affinity for resin; bind electrostatically
– Proteins of the same charge or different
strength of charge of resin: No good affinity
for resin; flow through column quickly, so
eluted first
• Result: protein similar to resin is held in
column
• To elute protein held to resin in column: use
buffer of higher ionic strength or stronger pH
– Changes ionic environment
– Ions in new (elution) buffer “exchange” for protein
(are more attractive to resin, so take the place of protein
on resin)
• Collect fractions from mobile phase + elution
buffer
• Test all fractions by activity assay
Affinity Chromatography
• Based on specificity of prot of interest for some
molecules to which it alone will bind
– Ex: Ab binds only specific Ag
• BUT binding must be reversible
• Solid support = specific binding molecule
(=ligand) covalently bound to beads, etc.
• Mobile phase = buffer of proper pH, ionic strength
to maintain activity of prot of interest
• Pack column; apply sample
• Begin slow mobile phase flow
• Prot of interest ONLY will bind to ligand
– Types of binding (must be reversible): ionic,
H-bonds, hydrophobic interactions
• To elute, may use solution of ligand
(competes w/ solid phase ligand) OR
buffers of diff strength, pH that disrupt
protein/ligand interactions
Electrophoresis
• Separation AND identification
• Based on overall charge of protein 
movement under influence of electric field
• Zone
– Semisolid or gelatinous medium (plate or slab)
– Spot protein mixture (w/ wanted + unwanted
proteins in solution) onto gel
– Apply electric field
• Molecules migrate toward anode (+ charged)
• Distance traveled dependent on charge, size of
protein
Most impt = size of protein
– Gel support acts as molecular sieve; smaller molecules
go faster toward anode, so migrate further
• Also, those more strongly charged move closer to
anode
• Use chemical to stain aa’s  bands representing
proteins of decreasing MW
• Run stds simultaneously
– Mixture of proteins of known MW; spot on one or
several lanes
– Electrophorese under same conditions as unknown
protein mixture
– Stain  “ladder” of bands (lowest to highest MW
proteins traveling some distance under these
conditions)
• Determine distance traveled
for each band from origin
• Plot x = distance migrated
for each std of known MW;
y = log MW of stds
• Yields std curve; find distance
traveled by unknown prot(s)
on curve; deter MW
• Can also cut gel, dissolve to
free proteins
Moving Boundary = IsoElectric
Focusing (IEF)
• Separation due to charge; based on isoelectric
pt of each prot
• Use gel of ampholytes (gel has regions of
different pHs)
• Spot sample in middle of gel
• Apply electric field
• Each prot in mixture will
migrate toward + or –
electrode, according to
charge
• Each prot will stop
moving when it reaches
pH region of gel = its
isoelectric pH
• Stain aa’s of prot’s w/
chemical
• Run stds simultaneously
– Mixture of proteins of known pI’s; spot in one or
several lanes
– Electrophorese under same conditions as unknown
protein mixture
– Stain  “ladder” of bands (lowest to highest MW
proteins traveling some distance under these
conditions)
– Determine distance traveled for each band from origin
• Plot x = distance migrated for each std of known pI
y = pH
• Yields std curve; can find distance traveled by unknown prot(s)
on curve  pI
Characterize wanted protein by
aa sequence
• Once wanted protein has been isolated from all
other cell molecules
• Old method: Break all peptide bonds  solution
of aa’s
• Analyze aa’s by chromatography
– Thin Layer Chromatography (TLC) – on coated plate or
paper support; various mobile phases separate aa’s from
each other
– High Pressure Liquid Chromatography (HPLC) – force
sample through small column packed with various
types of support; various mobile phases are forced
through column by high pressure pumps to separate
aa’s from each other
• Now have identified all aa’s in protein
• With original protein, use chem. rxn to label
amino terminal aa
– Use various enz’s to cleave prot at partic aa’s
along peptide chain  peptide fragments
– Analyze fragments for overlap; use knowledge
of all aa’s in protein  sequence
• New method: Automated
• Chem rxn to label amino terminal aa
• Cleave amino terminal aa
– Analyze for identity of last aa
– Rest of prot now has diff amino terminal aa (second to last
in original prot)
– Chem rxn to label second to last aa of amino terminal
– Cleave this terminal aa
– Analyze for identity of second-to-last aa
– Etc. etc. etc.
• Common now: identify gene for protein of
interest
– Isolate mRNAs (found w/in cell rich in wanted
protein) w/ gene nucleotide sequence
– Use mRNAs to identify gene
• mRNA will have complimentary sequence to gene
in DNA, so will pair in that region of DNA only
– Analyze gene for nucleotide sequence
– Use genetic code to determine aa sequence of
wanted protein from gene which codes for it