Experiment-4:
Densitometric analysis of proteins on Western blot
Objective: To quantify the amount of serum proteins present on a Western blot.
Theory:
To gain complete knowledge of the experiments defined in this website, it is important to
understand each of the following sections. Hence, we recommend that the content of each
section be read in the given order.

Sample collection and preparation: Serum proteins from blood samples of normal
participants and cancer patients are isolated and high abundant proteins are removed.

Protein quantification: The concentration of proteins in the sample is determined.
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SDS-PAGE: Serum proteins are separated on the basis of their molecular weights.

Western blotting: Proteins separated on the gel are transferred onto a PVDF membrane.
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Scanning of the blot: This provides the image needed to carry out analysis.

Densitometric analysis: This helps to identify the global expression pattern of protein
spots on the gel.
After the details of the technique are understood, the reader is encouraged to go through the
stimulations, protocols and manuals to get better insight of the process.
Many diseases manifest themselves through changes in the composition of serum proteins. One
of the main aims of clinical research is to quantify these changes and study their co-relation with
the progression of the disease. Proteomic strategies are being routinely used for the discovery
and identification of disease-specific protein markers from crude biological samples. Such
biomarkers could be effectively used for diagonistic purposes and could also provide targets for
the discovery of novel drugs.
The first step of a proteomic study is usually the separation of proteins using electrophoresis or
chromatography, followed by their detection and quantification. Western blotting is a commonly
used analytical technique to detect specific proteins in a given sample. This technique was
introduced by Towbin, et al, in 1979 and has been used extensively since then for protein
analysis. The first step in this technique is to separate the proteins using gel electrophoresis. The
separated molecules are then transferred or “blotted” onto a second matrix, usually a
nitrocellulose or polyvinylidene difluoride (PVDF) membrane. Details of this technique will be
explained in the following sections.
 Sample collection and preparation
Blood samples obtained from healthy participants and patients are incubated on ice for 30 min.
The blood coagulates and settles down as a pellet in the tube while the serum forms the dark,
yellowish, viscous supernatant. This crude serum sample is then subjected to mild sonication to
disrupt protein complexes and break the inter- and intra-protein interactions. 10% TCA-Acetone
is used to precipitate out the proteins from the resulting solution and the pellet is washed
thoroughly with ethanol. Serum proteins are then rehydrated using a rehydration solution
containing urea, CHAPS, DTT and TBP. Most of the proteins of diagnostic interest are present
at low levels in the serum. Since high abundance proteins like albumin could interfere in the
separation and detection of these low abundance proteins, they need to be removed from the
sample before carrying out electrophoresis. This is done by using commercially available serum
depletion kits. The kits contain affinity chromatography based columns which need to be
activated to charge the surface of the beads. Serum samples are then loaded onto the column and
incubated to allow the binding of the high abundant proteins to the charged surface of the column
beads., while flow-through contains the serum sample which is now rich in low abundant
proteins.
 Protein quantification:
It is important to know the concentration of protein sample being loaded onto an SDS-PAGE gel
so that similar quantities can be loaded onto subsequent gels and a comparison across the gels
can be made. It also helps in avoiding experimental artifacts and allows analysis of the gel in a
biological context. The Bradford method for protein quantification has been used in this
experiment. It is based on the principle of shift in absorbance maximum of the Coomassie
Brilliant Blue G-250 dye from 470 nm to 595 nm when it comes in contact with proteins. 
 SDS-PAGE:
SDS-PAGE brings about the separation of complex proteins by denaturing them into their
component polypeptides and resolving them on the basis of their molecular weights. . In SDSPAGE, polyacrylamide acts as a medium to support the separation while SDS is a strong anionic
detergent used to denature the protein. Molecular size of the protein is not considered in SDSPAGE. This is because SDS breaks the complexes into individual peptides and provides a
uniform negative charge to protein sample. Due to this, the intrinsic charge present on a protein
become negligible. During electrophoresis, proteins move towards anode due to negative charge
on them. Since SDS provides equal charge to mass ratio per unit protein, it causes the resolution
of proteins purely on the basis of their molecular weight
4. Western Blotting:
Western blotting is a simple, yet powerful technique, widely used in biological research for
detecting the presence of specific proteins. Once proteins are separated by electrophoresis on the
basis of their size, the separated molecules are transferred onto a nitrocellulose or PVDF
membrane. The membrane is blocked with albumin or milk proteins to prevent any non-specific
binding of antibodies to the membrane surface. The transferred protein is them complexed with a
primary or a secondary antibody which is labeled with an enzyme probe (Fig. 1). An appropriate
substrate is then added to the enzyme and together they produce a detectable product on the
membrane.
Fig.1: Schematic representation of the Western blotting procedure.
Apart from visual or colorimetic detections, one of the detection methods uses a
chemiluminescent substrate which reacts with the enzyme to produce light as a byproduct. This
light output can be captured using film, CCD camera or a phosphoimager that is designed for
chemiluminescent detection. Another detection method is the use of fluorescently tagged
antibodies, which are directly detected with the help of a fluorescence imaging system. All these
detection methods are designed such that they correlate with the abundance of the antigen on the
membrane. Hence the results achieved are easy to interpret, unique and unambiguous.
In addition to producing quantitative data about a protein, western blotting also provides
qualitative information related to that protein. Since proteins are separated by molecular weight
during the gel electrophoresis and then detected by a specifically directed antibody, this
technique confirms the identity of a target protein. Moreover, when data does not match the
expected results, this technique may provide clues as to what could be the possible reasons.
Protein degradation or cleavage or alternate splicing could be indicated by smaller than expected
bands, while bands seen at higher levels than expected could indicate increase in mass due to
glycosylation or multimer formation. Hence this technique is well suited for evaluating the
levels of protein expression in cells, and for monitoring fraction during protein purification. It is
also helpful for comparing expression of a target protein from various tissues, or seeing how a
particular protein responds to a disease or drug treatment.
The success of western blotting therefore depends on the successful transfer of the separated
proteins to the membrane and the specific detection of a target protein by appropriately matched
antibodies. Since antibodies are used to detect the target proteins, this technique is also called
Immunoblotting. Western blotting is also popularly used in combination with other, more
advanced techniques like 2-D gel electrophoresis, ELISA, immunohistochemistry or antibody
arrays. In such cases, western blots provide confirmation of the results obtained from such
techniques. Therefore, owing to its simplicity, this technique continues to be of immense value in
modern proteomic research.
 Scanning of the blot:
The blots are scanned using LabScan software version 6.0 (GE Healthcare). The blots are placed
in with proper orientation in the scanner, taking care that no air bubbles get trapped under the
blot.. An image of the blot is captured at 300 dpi and stored as a .tiff file with an appropriate
lable. A representative image is shown in Fig.2. Such images of blots can then be used for
comparison of the global expression profiling of proteins across different gels with the help of
commercially available software.
Fig. 3.: Image of serum proteins blotted onto a PVDF membrane.
 Densitometric analysis: 
Apart from molecular weight determination, western blotting can also be used to analyze the
differential proteins in multiple samples. In order to measure protein expression levels,
intensities of specific bands, corresponding to the proteins of interest are measured using
commercially available software. In this experiment, densitometric analysis of the blots was done
using Image Quant TL (IQTL) software (GE healthcare). The basic steps involved in this
analysis are as follows:1. Blot images are imported into the software and the contrast is adjusted such that the
bands are clearly visible on the blot image.
2. Area around each band is selected.
3. Background intensity is subtracted from the blot image.
4. Bands are then selected by drawing a tight boundary around them. Intensities of the
selected bands is then displayed in an excel format which can be exported for carrying
out further statistical analyses.