Protein Analysis

Protein structure
  DNA Proteins
genomic DNA-
chloroplast - different proteins: structural
types mitochonderia and functional ptoteins

Primary-Secondary -Tertiary -
Quaternary
structure doubel strand

units niclotides 4 amino acids 22

charge negative negative- positaive- neutal

base pair- killo base
Molecular mass pair dalton - killo dalton

difeer in size charge - size- structure

 Traditional Protein Analysis Techniques

three major protein analysis techniques:

Protein separation,
Western blotting
Protein identification (identification of amino
acids sequence and protein structure). We will
discuss only the first two types of them.
 Protein Separation
Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Protein Electro
sample Phoresis
Gel– Visualization
SDS Chemical specific for
separated sample (Protein)
Polyacrylamide
• It separating protein components based on mass when proteins
are separated by electrophoresis through a gel matrix, smaller
proteins migrate faster due to less resistance from the gel
matrix.
• Other influences on the rate of migration through the gel matrix
include the structure and charge of the proteins.
• can be used to estimate relative molecular mass, to
determine the relative abundance of major
proteins in a sample, and to determine the
distribution of proteins among fractions.
• The purity of protein samples can be assessed and
can be followed.
• Specialized techniques such as Western blotting,
two-dimensional electrophoresis, and peptide
mapping can be used to detect extremely scarce
gene products, to find similarities among them, and
to detect and separate isoenzymes of proteins. It is
an easy, inexpensive, and relatively accurate
manner.
 Gel Electrophoresis Steps
1. Preparing the samples for running
• Grinding the plant sampel in a suitabel buffer ----
centrifuge
• Heating 95C and add SDS+ loading dye ( Bromophenol Blue)
• SDS an anionic detergent, give an overall negative charge and
change its complex tertiary shape to create a long, rod-like
conformation .
• No secondary – tertiary –quaternary structure
 Polymerized acrylamide (Polyacrylamide) -2
• forms a mesh-like matrix suitable for
the separation of proteins of typical
size.
• Gel preparation requires casting two
different layers of acrylamide
between glass plates.
• Stacking gel, the upper layer (4% of
acrylamide)
• Running- Separating gel, the lower
layer (15% of acrylamide) is
responsible for actually separating
polypeptides by size. It is the actual
zone of separation of the
particle/molecules based on their
mobility.
• The gel concentration is inversely
proportional to the molecular
weight of proteins 
 Visualization of-3
,protein bands
• Visualizes the band under UV light
there are two Types of Stains:
• A. Coomassie Blue (Coomassie
Brilliant Blue) the traditional
method requires staining followed
by destaining to remove
background gel staining. It is the
most common and least sensitive,
Limited to ~100ng of protein.
• B. Silver Stain, Most sensitive test
of detection limit (0.1-1.0ng of
protein).
• C. staining band with Western blot
 Isozymes are protein markers
• Isoenzymes or isozymes are multiple forms of same
enzyme that catalyse the same chemical reaction
Different chemical and physical properties:
Electrophoretic mobility  Kinetic properties- Amino
acid sequence - Amino acid composition
• The technique is based on the principal that allelic
variation exists from many different proteins. For
example, alleles of malic dehydrogenase would both
perform the correct enzymatic function, but the
electrophoretic mobility of the two may differ.
Therefore, two alleles would not migrate to the same
location in a starch gel.
 Grouping of isozymes
Isozymes are divided into three categories
depending on the way they are biosynthesized:
• 1. isoenzymes (multilocus isozymes) arise from
multiple gene loci, which code for structurally
distinct polypeptide chains of the enzyme;
• 2. allozymes (or alleloenzymes), are structurally
distinct variants of a particular polypeptide
chain, coded by multiple alleles at a single locus;
• 3. secondary isozymes result from post-
translational modifications of the enzyme
structure;
• The procedures to identify isozyme variation is simple.
• A crude protein extract is made from some tissue sources,
usually leaves.
• The extracts are next separated by electrophoresis in a starch
gel.
• The gel is then placed in a solution that contains reagents
required for the enzymatic activity of the enzyme you are
monitoring.
• In addition, the solution contains a dye that the enzyme can
catalyze into a color reagent that stains the protein. In this
manner allelic variants of the protein can be visualized in a gel
• The separation of isozymes on the basis of
surface charge (and to a lesser extent on
molecular weight) may be achieved by 
electrophoresis in starch gel, acrylamide gel, 
agarose, cellulose acetate or Cellogel under
conditions of pH, ionic strength, and ionic
composition appropriate for a specific
enzyme.11 (See also Section XIV.)
Enzyme-linked immunosorbent assay (ELISA)
• The enzyme-linked immunosorbent assay (ELISA) is an
immunological assay commonly used to measure antibodies,
antigens, proteins and glycoproteins in biological samples. Some
examples include: diagnosis of HIV infection, pregnancy tests, and
measurement of cytokines or soluble receptors in cell supernatant
or serum.
• ELISA assays are generally carried out in 96 well plates, allowing
multiple samples to be measured in a single experiment. These
plates need to be special absorbant plates (e.g. NUNC Immuno
plates) to ensure the antibody or antigen sticks to the surface. Each
ELISA measures a specific antigen, and kits for a variety of antigens
are widely available.
• The ELISA pictured in Figure 1 is what is known as a sandwich
ELISA, here two sets of antibodies are used to detect secreted
products, e.g. cytokines. The method is stepwise in the order
shown.
• The 1st step is to coat the ELISA plate with capture antibody, any
excess, unbound antibody is then washed from the plate. The
capture antibody is an antibody raised against the antigen of
interest.

• Next the sample (e.g. urine, serum, or cell supernatant) is

added. Any antigen found in the sample will bind to the
capture antibody already coating the plate. Again any excess
sample is washed from the plate.

• In step 3, detection antibody is added. This antibody is labelled

with an enzyme, usually horse radish peroxidase or alkaline
phosphatase. Detection antibody binds to any target antigen
already bound to the plate. Finally, a substrate is added to the
plate. ELISA assays are usually chromogenic using a reaction that
converts the substrate (e.g. TMB or ABTS) into a coloured
product which can be measured using a plate reader.
 western blot
• Blots are techniques for transferring DNA (Southern Blot),
RNA (Northern Blot) and proteins (western blot)

• western blot : used tool to identify and quantify a specific

protein in a complex mixture (size and amount). It used also in
disease diagnosis where it can detects antibody against virus
or bacteria in serum, and useful to detect defective
proteins.

• low cost, sensitivity, and flexibility

• a popular technique in the life sciences as a convenient and

reliable research tool. This method is used in the fields of
molecular biology, biochemistry, immunogenetics and other
molecular biology disciplines.
• rely on the specificity of binding between a
molecule of interest and a probe (is typically
an antibody) to allow detection of the
molecule of interest in a mixture of many
other similar molecules.
• only one band should be visible.
• The thickness of the band corresponds to the
amount of protein present.
 Procedure

1. Extraction of protein
2. Gel electrophoresis: SDS PAGE
3. Blotting: electrical or capillary blotting
4. Blocking: BSA
5. Treatment with primary antibody
6. Treatment with secondary antibody( enzyme labelled
anti Ab)
7. Treatment with specific substrate; if enzyme is alkaline
phosphatase, substrate is p-nitro phenyl phosphate which
give color.
 steps
• 1-Tissue Preparations and
Gel Electrophoresis

• 2-Transferring- Blotting
• In order to make the
proteins accessible to
antibody detection, they
are moved from within the
gel onto a membrane made
of nitrocellulose or
polyvinylidene difluoride
(PVDF).
 Blocking and Antibody Incubation -3

• Blocking: The membrane has the

ability to bind to proteins in in
this case both the target and
antibodies are proteins
• it prevents antibodies from
binding to the membrane
nonspecifically. It is achieved by
placing the membrane in a dilute
solution of protein - typically
Bovine serum albumin (BSA).
• Incubation of antibody
 4-Detection –Quantification:
During the detection process, the
membrane is "probed" for the
protein of interest with a modified
antibody which is linked to a
reporter enzyme, (such as
horseradish peroxidase (HRP)), which
when exposed to an appropriate
substrate drives a colorimetric
reaction and produces a color. This
signal is captured on a film which is
usually developed in a dark room.