July 12, 2013 [ Biochemistry: ANALYSIS OF PROTEINS

LECTURER: Dr. Noel S. Quiming o Molisch - sugar backbone in

mitochondrial DNA
ANALYSIS OF PROTEINS o Biuret - mitochondrial
3 major steps: membrane transport
1. Isolate proteins from the source proteins; mitochondria
2. Analyze the proteins makes proteins
3. Determine the amino acid o Sudan - has a mitochondrial
sequence membrane composed of
lipids (lipid bilayer)
Differential Centrifugation o Schiff's - contains
 method of separating subcellular mitochondrial DNA
components using application of 3. Microsomal fraction
different forces  from fragments of the endoplasmic
 depends on differences in densities of reticulum
the subcellular components  smallest particles and highest
Homogenization centrifugation force
 done before centrifugation; breaking of  positive for all tests because..
cells for the release of macromolecules o Molisch -contains soluble
 grinding a sample in a buffer solution carbohydrates
(buffer dissolves the proteins) o Biuret - protein synthesis
4 Tests: occurs in rough ER (rRNA
1. Molisch - test for carbohydrates; in ribosomes)
gives a purple ring o Sudan - fatty acid synthesis
2. Biuret - test for presence of occurs in smooth ER
peptide bonds (proteins); gives a o Schiff's - rough ER contains
violet solution ribosomes with RNA
3. Sudan - Sudan dye is lipid soluble,
*Why Sudan test sometimes exhibits a
gives a red solution
4. Schiff's - test for nucleic acids; false negative?
gives a pink solution  When amount of lipid is small in
proportion to aqueous buffer, Sudan IV
3 Cellular fractions (in order): flocculates.
1. Nuclear fraction
 separated first and at lowest **Last supernatant liquid contains the
centrifugation force because it has soluble proteins and glycogens so it is
the largest particles kept for isolation of these components
 positive for all tests because..
o Molisch - sugar backbone in Methods of Protein Isolation
DNA and RNA 1. Salting out
o Biuret - DNA is conjugated to  solubility based; salt is added in an
histone proteins aqueous medium, competes with
o Sudan - has a nuclear protein, salt is more soluble so the
membrane composed of lipids protein salts out or precipitates
(phospholipid bilayer)  can be used to separate different
o Schiff's - nucleus contains DNA proteins because salt
(deoxyRIBONUCLEIC ACID) concentration at which they
and RNA (riboNUCLEIC ACID) precipitate vary (e.g. 0.8 M
2. Mitochondrial fraction ammonium sulfate precipitates
 requires higher centrifugation fibrinogen, 0.24 M is needed to
force precipitate albumin)
 positive for all tests because..

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 July 12, 2013 [ Biochemistry: ANALYSIS OF PROTEINS
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 Larger proteins - only have

access to mobile phase in
between beads so they are
2. Dialysis eluted first; consumes smallest
 proteins separated based on amount of mobile phase
particle size; use of a semi-  Application of gel-filtration: can
permeable membrane immersed in be used in determination of
buffer approximate MW of unknown
 molecular cutoff- the lowest proteins by comparing with
molecular weight of protein that is proteins with known MW
retained in the membrane (lower
than that, proteins will diffuse out,
higher MW proteins are retained)
3. Liquid Chromatography
 Review of paper
chromatography:
o stationary phase: water
adsorbed in filter paper;
mobile phase: solvent
o based on affinity of solute
to stationary and mobile
phase; proceeds slowly if
solute has high affinity for
stationary phase and vice
versa
 Chromatography is classified
based on the polarity of stationary
phase
o Normal Phase: stationary
phase is polar, mobile is
nonpolar
o Reversed Phase: stationary
phase is nonpolar, mobile is
polar

 Types:
a. Size-exclusion chromatography
 basis is molecular weight
 Gel-filtration chromatography -
stationary phase= polymer
beads with pores of different
sizes; mobile phase= found
inside and in between beads
 Smaller proteins - fit inside
pores of beads; have access to
mobile phase inside and in
between beads so they are last
to elute

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b. Ion exchange chromatography

 separation based on charge
 Stationary phase = anion exchanger (+ charged) or cation exchanger (-
charged), usually made of resins
 The stationary phase attracts a specific charge of protein so the opposite charge
is eluted out.
 To get the proteins attracted/ still bonded to column a mobile phase with a
cation of greater affinity to the stationary phase is used
 Also affected by size (provided same net charge): biggest proteins are eluted
first
 Same size but different net charges: lowest net charge is eluted first
c. Reversed phase chromatography
 just like ion-exchange but matrix contains aliphatic chains
 based on hydrophobicity: the more hydrophobic proteins bind to the matrix
 based on polarity, more polar ones are eluted first
 proteins are polar so there is less affinity to the nonpolar stationary phase
 stationary phase is nonpolar, mobile phase is polar
d. Affinity chromatography
 stationary phase: contains a specific ligand, a molecule that binds to another
molecule (specific interactions like antibodies to antigens)
 proteins with high affinity to the stationary phase will be trapped, those with no
interaction will be eluted out
 the target protein will be retained inside the column so a mobile phase
containing the ligand is used (the column will be saturated with a solution
containing the ligand so protein can be eluted out)

GEL ELECTROPHORESIS ◊ Electrophoretic mobility depends

Electrophoresis consists in the migration inversely on the friction coefficient.
of proteins through matrix (porous gel) in Separation of proteins is based on 2
response to an electric field. factors:
- use of current/electricity to 1. charge
separate proteins 2. size - because the gel used can act
● Proteins move because they are as a sieve/filter
charged base on their amino acid
components. ◊ 2 electrodes:

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Cathode – negatively charged  The SDS will interact with the protein
electrode where the through hydrophobic interaction of
CATIONS migrate nonpolar tail. The protein will be coated
Anode – positively charged with SDS – therefore, all proteins will
electrode where possess negative charges.
ANIONS migrate

 The matrix is typically a crossed-linked

polymer (often polyacrylamide).
Inside the chamber → gel (usually
transparent)
 Place mixture of proteins and apply
current/electricity. Once current is
applied, the proteins will move base
on their charges.
 At the bottom of the chamber is the
positively charged electrode →
negatively charged protein will
migrate towards the positive
electrode.

 Proteins with same size, different

net charges
o the bigger the charge, the
faster the migration
Proteins with same net charge,
different sizes
o the smallest protein would
migrate the fastest

 To eliminate charge as a factor:

SDS-PAGE (Sodium Dodecyl Sulfate

Polyacrylamide Gel Electrophoresis)

Protein acrylamide gel electrophoresis is

performed in the presence of the
detergent sodium dodecyl sulfate (SDS-
PAGE). Detergent has negative charge
and binds to all proteins with similar
affinity – protein binds SDS proportionally
to their size.

Sodium Dodecyl Sulfate is an example

of an anionic surfactant – contains a polar
head and a nonpolar tail. (the polar head
is negatively charged)

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The negatively charged SDS (sodium dodecyl sulfate)-protein

complexes migrate in the direction of the anode, at the bottom
of the gel.

 Separation will be based only on the molecular size/weight of

protein – the smallest moving most rapidly. The larger
proteins will have more interaction with the matrix causing
them to slow down.

 How to detect proteins in gel:

Proteins are visualized by staining them with a non-specific dye.
●dye – proteins will be stained wherein 1 band = 1 protein
▪silver nitrate

 Determining the approximate molecular weight of a protein:

standard proteins – also called molecular marker/ladder; proteins with known
molecular weight
smallest protein migrates first

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Plot of molecular weight vs. the relative migration/time

Mobility is inversely proportional to the logarithm of the
molecular weight of the protein.

ISOELECTRIC FOCUSING

▪ Electrophoresis is performed in a matrix with a pH gradient

→gel contains a pH gradient from cathode to anode –
decreasing pH
▪ Separation of proteins is based on their isoelectric point (pI) – pH at which the protein
has a net 0 charge or neutral charge (electrically neutral)

 A stable pH gradient is established in the gel after application of an electric field.

 Protein solution is added and electric field is reapplied.
 Proteins will migrate towards the anode.
 After staining, proteins are shown to be distributed along pH gradient according to
their pI values.
→ from cathode to anode – decreasing pI
 Net charge of protein on top of gel with greater pH – negative charge –
because all ionizable groups will be completely deprotonated
 Once the isoelectric point of a particular peptide is reached, that peptide
will become neutral – therefore, it will no longer migrate. Others will
migrate until they reach their isoelectric point.

TWO DIMENSIONAL GEL ELECTROPHORESIS

→combination of 2 techniques
1st dimension – isoelectric focusing
2nd dimension – SDS-PAGE

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DENSITY GRADIENT CENTRIFUGATION

→proteins are separated based on their densities (specific gravity)

●This process uses a solution with different viscosities.

Example: sucrose / solution of cesium chloride
 Densities depend on the concentration of the sucrose solution.
(increasing concentration – increasing viscosity)

The steps are as follows:

(1) Form a density gradient.
→ formulate sucrose solution of different concentration and pour it in a test
tube
→ the most concentrated sucrose solution will have the highest density
(2) Layer the sample on top of the gradient.
→Without shaking to prevent mixing of sucrose solutions
(3) Centrifuge the tube.
→Top to bottom – increasing densities
→ Proteins with the same density as the sucrose solution will be at the same
level
(4) Puncture tube at the bottom and collect the samples.

Matrix-Assisted Laser-Desorption Time-of-Flight (MALDI-TOF) Mass Spectrometry

- proteins are first adsorbed in a matrix
- matrix – can be composed of a polymer/any material
(1) The protein sample, embedded in an appropriate matrix, is ionized by the
application of a laser beam.
 Expose matrix to a laser beam – proteins will be desorbed out of the matrix
(2) An electrical field accelerates the ions formed through the flight tube toward the
detector.
 Once proteins are already charged, they will pass through the part of the
machine where ions are deflected base on their charges.
 The lightest ions arrive first
(3) The ionizing laser pulse also triggers a clock that measures the time of flight (TOF)
for the ions.
 The time it takes for the protein to travel is measured by the instrument.
 Both charge and size is considered in the separation
→smaller protein is less dense, lighter, moves faster

SPECTROPHOTOMETRIC ANALYSIS
- used for protein quantization
- enzyme kinetics
A spectrophotometer is used to measure the absorbance of a protein sample.
 Measures the absorbance of the sample
 Absorbance can be used to estimate the ratio between protein and nucleic acids in
a protein preparation.
 Absorbance can be used to estimate the total amount of protein in a mixture of
proteins.

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 Absorbance permits to measure protein concentration of a pure protein sample for

which the amino-acid sequence is known.
→ Absorbance is related to the concentration of either the substrate of the enzyme or the
product of the enzyme catalyst reaction.

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