Transcript proteins - Portal UniMAP

PTT 202:
ORGANIC CHEMISTRY FOR BIOTECHNOLOGY
PREPARED BY:
NOR HELYA IMAN KAMALUDIN
[email protected]
Introduction of Proteins
Proteins are made up of about 22 amino acids,
which are linked by the peptide bond.
Methods for the quantitation of proteins are either suitable for
all proteins or designed to measure individual proteins.
Such specific methods may depend on either a
preparative stage in the analysis or the use of a specific
characteristic of the protein.
Separation of Proteins
Immunological
methods
Immunoaffinity
purification
Electrophoresis
Precipitation
Separation
of proteins
Chromatographic
methods
Precipitation
High concentrations of a variety of salts including sulphates, sulphites
and phosphates can be used to precipitate proteins
• But, each fraction produced still consists of a mixture of protein
• Usually requires further purification.
• Excessive denaturation of the protein is avoided by the use of low temperatures.
Salt fractionation techniques prepare protein fractions by successively
increased concentrations of the salt.
Various alcohols may also be used and the classical Cohn fractions of
serum proteins are separated using specific concentrations of ethanol
under carefully controlled conditions of temperature and pH.
Electrophoresis
Electrophoresis has a major application in the separation of proteins because
charge and molecular size are important to both electrophoretic separation
and to protein structure.
Conventional techniques: Use a flat supporting medium, e.g. cellulose acetate
strip or open gel plate
Capillary techniques: Instrumental technique and is very dependent upon the
availability of commercially produced equipment and
reagents.
• In both techniques, the key factors in the separation of proteins are the choice
of the operating pH and the electrophoretic conditions to be used.
The technical decisions are largely made by the manufacturer rather than analyst.
• It is essential, however, that the analyst can identify the molecular features of
the analytical problem and then select an appropriate technique.
Technical aspects of electrophoresis
Supporting
medium
The
methods of
capillary
electrophore
sis
performed
Concentrati
on of buffer
Iso-electric
focusing
technique
Factors affecting
electrophoresis
pH of buffer
1. Supporting medium
Filter paper
• Has significant disadvantages, the most serious being the adsorption of
proteins.
• Modified cellulose media such as cellulose acetate shows significantly less
adsorptive effects.
• But, together with a uniform pore structure, it result in a greatly improved
resolution.
Starch and polyacrylamide gels
• Show improved resolution due to a molecular sieving effect.
Patterns of separation for different media
• It is important to realize that the use of these different media for the same
sample will result in separation patterns that cannot be easily compared
with one another.
• The separation of serum protein on cellulose acetate will result in 5-7
bands, while the use of polyacrylamide gel will give 17 bands.
2. Iso-electric focusing technique
Iso-electric focusing technique
• Probably give the best resolution and many of the
resulting bands are due to specific proteins.
• They are used mainly as a qualitative or a semiqualitative technique due to:
• The large number of bands that develop
• Particular value when successive samples from the
same source need to be compared for the presence or
absence of a particular protein or for investigation of
physical properties.
3. Buffer pH
pH of the buffer
• It may affects the charge carried by the protein.
• In theory any pH may be used, but, in practice pH
values greater than the iso-electric pH of the protein
give better separation than others pH values.
• In selecting the conditions for the separation of a
particular protein mixture, a buffer pH that gives the
greatest difference in the charge carried by each
individual protein results in the greatest difference in
velocities and hence in the final distances moved.
• In practice buffer of pH 8.6 are the most frequently
used.
4. Buffer concentration
Concentration of buffer
• Could also affect the mobility of protein.
• At high concentration, the zeta potential of the
protein is reduced resulting in a shorter distance of
migration.
• However, because higher concentrations of buffer
give improved resolution, a compromise
concentration has to be found and buffer with ionic
strength (μ) varying from 0.025 to 0.075 are
frequently used.
5. The methods of capillary
electophoresis performed
Capillary electrophoresis
• The various types of capillary electrophoresis are
performed either in free solution or in gels.
• The choice of method depends on the nature of the
sample and the analytical objectives.
• However, capillary electrophoresis including isoelectric focusing and SDS electrophoresis is
particularly useful for protein applications.
Quantitative aspects for electrophoresis
Quantitative
aspects
Semiquantitative
method
Alternative
quantitative
method
1. Semi-quantitative method
Conventional electrophoresis
• In conventional electrophoresis, semi-quantitative method refer
to the amount of dye bound in the pores of the supporting
medium which offer directly correlated to the amount of
protein.
• Various fractions usually being expressed as a percentage of the
total rather than in absolute amounts.
• The amount of dye bound by each fraction can be simply
determined by cutting out the stained bands and eluting the dye
into a fixed volume of a suitable solvent.
• The absorbance of each solution is measured and the sum of
absorbance values assumed to be proportional to the total
amount of protein.
• Hence the amount of protein in each fraction can be calculated
as the percentage of the total.
2. Alternative quantitative method
Densitometer (modern electrophoresis)
• More satisfactory method of quantitation by scanning the
stained electrophoretic strip using a densitometer, which is a
modified photometer in which the electrophoretogram
replaces the usual glass cuvette.
• The strip is slowly moved across the light path and the signal
from the photoelectric detector is plotted by a pen recorder.
• The result is a trace that plots the absorbance value against
the distance along the electrophoretogram and the area
under the trace is proportional to the total protein content.
• From the area under each peak, the proportion of protein
associated with that peak can be calculated.
Immunological methods
Immunoblotting technique
(Western blotting)
• Immunoblotting technique use antibodies (or
other specific ligands) to identify target
proteins among a number of unrelated protein
species.
• The prerequisite is the availability of an
antibody, either polyclonal or monoclonal,
against the test protein.
The steps of immunoblotting
After the initial separation by a conventional
electrophoretic technique in a gel, the proteins are
transferred (or blotted) electrophorecally from the gel to a
membrane, usually nitrocellulose.
The gel and the membrane, which has been previously
soaked in a suitable electrophoretic buffer, are then
sandwiched between two electrodes.
A voltage is applied, e.g. 100 V, and the proteins migrate
from the gel to the adjacent membrane.
After about 1 h, the membrane is removed and carefully
washed with buffer and a dilute solution of bovine
albumin to block any subsequent, non-specific
adsorption of antibodies to the membrane.
The steps of immunoblotting (cont)
The next step involves treating the membrane with a
suitable dilution of the specific antibody and allowing the
reaction to take place for at least 1 h.
Excess antibodies are then washed from the membrane
and the bound antibody which remains is detected using
a second antibody against the first, e.g. anti-rabbit
immunoglobulin.
The bands can then be visualized using an appropriate
method.
The resulting patterns of zones is then compared with the
electrophoretogram prior to immunoblotting and the
specific proteins pinpointed for any further investigation
or separation.
Immunoaffinity purification
Useful technique for the separation and purification of individual
proteins due to the powerful immunogens and the availability of
specific antibodies.
Has been used to purify a wide range of proteins such as
hormones, membrane receptor and complement
proteins.
The availability of suitable antibodies is essential and these may
be raised by whole animal polyclonal techniques or by
monoclonal cell culture.
The former antibodies may need some prior
purification before being immobilized.
Chromatographic methods
Various chromatographic techniques may be applied to the
study of protein mixtures.
Gel permeation chromatography is frequently used to separate protein
mixtures but it is necessary to have some prior knowledge regarding the size
of the proteins present in order to select the most suitable gel.
Ion-exchange chromatography using the substituted cellulose ionexchangers is frequently used in the preparative aspects of protein
analysis.
Affinity chromatographic techniques, including those that employ
antibodies as ligands, permit highly specific separations of proteins.
Reverse-phase HPLC can be used for the separation of peptides
and proteins.
Hydrophobic interaction chromatography (HIC) is particularly useful for
protein separations.
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