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Experiment-4 : Densitometric analysis of proteins on western blots
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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 abundance proteins are removed.
·      Protein quantification: The concentration of proteins in the sample is determined.
·      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.
·      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.
 
1.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.
 
2. Protein quantification:
It is important to know the concentration of protein sample being loaded onto a 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. 
 
3. 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 SDS-PAGE, 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 SDS-PAGE.  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 becomes negligible. During electrophoresis, proteins move towards the anode due to negative charge on them. Since SDS provides equal charge to mass ratio per unit protein, it results in 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 then 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 colorimetric 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 a 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 the 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.
1.    Scanning of the blot:
The blots are scanned using LabScan software version 6.0 (GE Healthcare).  The blots are placed in 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 label.  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 softwares.
 
 
Fig. 2: Image of serum proteins blotted onto a PVDF membrane.
 
 
2. 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.

 

 

 

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