Experiment-3: Densitometric analysis of proteins on SDS-PAGE gels Objective: To quantify the amount of serum proteins resolved by SDS-PAGE gels.
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: Method of drawing blood sample and separating the serum fraction is described.
• Sample preparation:Proteins from the serum sample are precipitated and high abundant 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.
• Staining the gel: Pattern of protein separation can be visualized after staining the gel.
• Scanning of the gel: This provides the image needed to carry out analysis.
• Densitometricanalysis: 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.
One of the principle areas of biological research focuses on the identification of specific markers for quick and confirmative screening of different diseases. After the successful completion of the human genome project, the proteomic profiling of various disease states has gained new importance. Proteomics has made significant impact on many aspects of clinical research like the understanding of disease pathogenesis and the discovery of novel biomarkers for diagnosis of a disease. Early detection of some fatal diseases like cancer and autoimmune disorders can be achieved by determining the protein expression changes in different biological fluids. Blood is an easily accessible body fluid, which contains a wealth of information regarding the pathophysiological condition of an individual person.The liquid portion of blood is referred to as plasma, and the removal of fibrinogen and other clotting factors from plasma results in serum. This serum contains a dynamic range of proteins, electrolytes, waste products, immunoglobulins, dissolved gases and water. It can also show the presence of a variety of proteins released by diseased tissue, making it an attractive sample for clinical studies. Expression pattern of various serum proteins undergoes rapid alterations in response to internal or external stimulus. Hence, a correlation can be established between the serum protein levels and the progression of the disease. However, due to the presence of wide dynamic range of proteins and their low abundance, identification of disease-specific protein biomarkers in serum is very challenging. Successful detection of these low-abundance disease biomarkers depends on the development of novel methods which can separate low-abundance proteins from the high-abundance proteins and robust detection techniques which have high sensitivity and specificity. One such technique is the Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), a high-resolution one-dimensional technique which is suitable for analytical resolution of complex protein mixtures.
1. Sample collection: Observance of proper procedures during the collection of blood and separation of serum is of utmost importance since it influences the result and reproducibility of the proteomic experiments. Proper care during sample handling prevents the denaturing of proteins in serum. Blood samples are drawn from the antecubital vein of healthy participants and patients suffering from a certain disease. The tubes that are used for collecting blood do not contain any external anti-coagulating agent. Generally, Vacutainer tubes are used to bring about efficient and rapid withdrawal of blood. Immediately after blood collection, the tubes are place on ice for 30 min. Blood coagulates and fibrin clots form during this incubation time, which is essential for the separation of serum. The coagulated blood is then centrifuged at 2500 rpm at 20 °C for10 min. Blood clot containing the different types of blood cells and clotting factors forms the pellet while the serum forms the dark, yellowish, viscous supernatant. Without disturbing the pellet, the supernatant is carefully aspirated out using a micropipette and collected into a fresh, clean, labeledmicrocentrifuge tube. Usually about 1.5 to 2 mL of serum is obtained from 5 mL of whole blood. Multiple aliquots of each serum sample are made and dispensed into appropriately labeled tubes. The serum containing tubes are then stored at −80 °C until further use.
2. Sample preparation: Though serum is devoid of any blood cells and clotting factors, it still contains a wide variety of extremely complex proteins, which can hinder the separation on SDS-PAGE. Various physical and chemical methods have been developed which can reduce the complexity of the crude serum sample and improve the quality of protein extraction. Sonication is one such method, which produces high frequency sound waves to disrupt cell complexes and the inter- and intra-protein interactions.Crude serum samples are subjected to mild sonication in the presence of ice, to absorb the heat that is generated during the process. 10%TCA-Acetone is then used to precipitate out the proteins from the resulting solution which may contain lipids, detergents and other impurities. The precipitated proteins are collected by centrifugation and washed with ethanol to remove any organic contaminants that may be present. Proteins are then rehydrated using a rehydration solution containing urea, CHAPS, DTT and TBP. Urea is a chaotropic agent and helps in stabilization and unfolding of proteins so that the charged surfaces of the protein are exposed towards the solution. CHAPS, i.e. 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, is a zwitterionic detergent used for solubilizing proteins.Dithiothreitol (DTT) efficiently reduces inter and intra molecular disulphide interactions, thereby exposing the ionic surfaces of the proteins. Tributylphosphine, or TBP, used in the solution, is a reducing agent which increases solubility of the protein. Since high abundance proteins could interfere in the separation and detection of the low abundance proteins, they need to be removed from the sample before applying it to an SDS-PAGE gel. This is done by using commercially available serum depletion kits containing affinity chromatography based columns along with the relevant buffer solutions and collection tubes. A column is first activated by using the specified amount of activation buffer provided in the kit. This is done to charge the surface of the column. The serum sample is thenloaded onto the column and incubated to allow the binding of the high abundant proteins to the charged surface of the column beads. The column is then centrifuged with a fresh collection tube attached to the outlet. The collected flow-through contains the serum sample which is now rich in low abundant proteins.
3. 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 method used for protein estimation should be such that its reagents are compatible with the chemicals present in the sample solution. The standard range is set to a higher extent, in order to accommodate the absorbance reading for unknown samples. The process is carried out in duplicates for standards and samples to get accurate result outputs. Inthis experiment, Bradford method has been used. It is based on the principle of shift in absorbance maximum of the dye in the presence of proteins. The Bradford reagent contains Coomassie Brilliant Blue G-250 dye, which shows a brownish-red color in the unbound state and has an absorbance maximum of 470 nm. It binds to the proteins with non-covalent interactions like electrostatic and Van der Waal’s forces to form a blue colored complex, having absorbance maxima of 595 nm.
4. SDS-PAGE: Separation of complex proteins is best brought about by denaturing them into their component polypeptides. These polypeptides can then be separated from each other using various techniques. Amongst them, SDS- PAGE [Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis] is a widely used.It separates proteinsaccording to their electrophoretic mobility based on molecular weight and charge. In SDS-PAGE, polyacrylamide acts as a medium to support the separation while SDS is a strong anionic detergent used to denature the protein. This technique follows the principle of electrophoresis i.e. the separation of charged molecules (macromolecules having charge) in applied electric field. The separation of the proteins depends on many factors including buffer strength, size of the molecule, charge, percentage of the gel composition and viscosity of the sample. Polyacrylamide gels restrain larger molecules from migrating as fast as smaller molecules. In a gel of uniform density the relative migration distance of a protein (Rf, the f as a subscript) is negatively proportional to the log of its mass. The velocity of a charged particle moving in an electric field is directly proportional to the field strength and the charge on the molecule and is inversely proportional to the size of the molecule and the viscosity of the medium.If proteins of known mass are run simultaneously with proteins whose mass is unknown, the relationship between Rf and mass can be plotted, and the masses of unknown proteins estimated. Hence, the relative migration of the proteins in SDS-PAGE of uniform density is directly proportional to field strength, charge on the molecule whereas inversely proportional to molecular weight of the protein. Molecular size of the protein is not considered in SDS-PAGE. This is because SDS is a strong anionic detergent which can break the complexes into individual peptides and denature the proteins. Furthermore, heat treatment leads to breakage of non-covalent bonds, while β-mercaptoethanol or DTT treatment breaks the di-sulfide bonds. The final product after treatment is more or less the complete primary structure of the protein, with a more or less linear shape.
Fig. 1: Schematic representation of the steps involved in sample preparation before it is loaded onto an SDS-PAGE for separation.
4A) Chemicals used in SDS-PAGE gel
Acrylamide (C3H5NO; MW: 71.08) It is a white odourless crystalline solid,easily soluble in many solvents such as water, alcohol, ether and chloroform. It is essential to store acrylamide, a neurotoxin, in a cool dark and dry place to reduce autopolymerisation and hydrolysis.Acrylamide monomers get polymerized via vinyl addition polymerization and the reaction is accelerated by free radicals. These linear polymers further interlinked together in presence of bis acrylamide and form a gel. This interlinked polymerized gel is a matrix like structure that retards the migration of the proteins as a function of their size.The ratio of acrylamide to bis-acrylamide in part determines the "pore size” of a gel.
Bisacrylamide (N, N’-Methylenebisacrylamide) (C7H10N2O2; mW: 154.17) Bisacrylamide is the most frequently used cross linking agent for polyacrylamide gels. Bisacrylamide can crosslink two polyacrylamide chains to one another, thereby resulting in a gel.
Sodium dodecyl sulphate (SDS) Sodium dodecyl sulphate (SDS) is an anionic detergent which provides a uniform negative charge to protein sample with an even distribution of charge per unit mass i.e 1.4 g of SDS/gm of protein. Due to this, the intrinsic charge processed by a protein become negligible. SDS denatures the proteins as well as complexes into constituent polypeptides. 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 (Fig. 2)
Fig.2: Action of SDS and heat treatment on the structure of the protein
Ammonium persulfate (APS)(N2H8S2O8; MW: 228.2) APS is an oxidizing agent and is a source of free radicals. During polyacrylamide gel preparations, APS play vital role along with TEMED to catalyze the polymerization by generating free radicals. It is light sensitive chemical often stored in dark bottle. It is freshly prepared at the time of gel casting.
Tetramethylethylenediamine (TEMED)(C6H16N2; MW: 116.21) TEMED is the important component required for polymerization. Its major role is to stabilize the free radicals and support the polymerization. The time required for polymerization and turbidity of the gel depends on the free radicals, and hence, indirectly depends on TEMED concentration. Generally APS and TEMED are used at approximately equimolar concentrations in the range of 1 to 10 mM.
Counter ions like glycine maintain a balance of the intrinsic charge of the buffer ions and also affect the electric field strength during electrophoresis. During electrophoresis, chloride ions moves towards anode because of their smaller size and high negative charge, and the glycine ions, being protonated at pH 6.8, are retained at cathode. The proteins then move between the chloride and glycine because they are larger than chloride ions, and more negatively charged (due to SDS) than the protonated glycine. Glycine has been used as the source of trailing ion because itspKaof 9.69 and mobility are such that the effective mobility can be set at a value below that of the slowest known proteins of net negative charge in that pH range.
4B) Sub-parts of SDS-PAGE gel SDS-PAGE gels are discontinuous gels, composed of two parts, viz., stacking and resolving gels. These gel are set one above the other and differ from each other in terms of their composition and pH . The running gel has a pH of 8.8 with HCl while that of the stacking gel is 6.8.
Laemmle buffer or loading buffer
Laemmle buffer or loading buffer is a combination of SDS, Glycerol, β-mercaptoethanol and bromophenol blue (BPB). Samples which are ready to load on a gel is mixed with Laemmle buffer and heated on heat block at 95° C for 5 min. The specific role of each component of the Laemmli buffer is as follows:
SDS: Denatures the protein and breaks the protein complexes into peptides
Glycerol: Stabilizes the protein. It also provides viscosity to the sample to retain it in the well during loading and prevent the dispersion of the protein sample.
β-mercaptoethanol: Oxidize the cysteine residues and break the disulfide bonds to linearize the protein molecule.
BPB: A low molecular weight, highly mobile, negatively charged blue dye. It acts as tracker during protein separation. Additionally, heating of the protein sample leads to denature of the protein and also promote the binding of SDS to the protein.
Running buffer or SDS-PAGE buffer
This buffer provides a mobile front and conductivity to the electrophoretic apparatus.The widely used running buffer consists of glycine, tris-HCl and SDS. It provides ions which can promote the migration of proteins in gel towards anode,baseded on molecular weight. The selection of buffer is very important for protein separation because the buffer components should not react with the proteins during running.
Glycine is an amino acid having week acidic nature. It exists in different states based on pH of the system. In the low pH region, it has positive charge. As pH increases, it becomes uncharged and at high pH it has negative charge. pH of running buffer is around 10 due to presence of Tris-HCL. Hence glycine has a net negative charge in this solution. When an electric field is applied, the negatively charged glycine molecules start moving towards anode by entering into the stacking gel. The pH of the stacking gel is 6.6. This leads to significant decrease in charge on glycine which leads to a slowdown in its movement. On the other hand, chloride ions having negative charge start moving fast towards anode. The slow moving glycine ions and fast moving chloride ions make a narrow zone of pH gradient. As a result, the SDS-bound proteins form a sharp band between these two ions and move forward as a sharp band till theyreach the resolving gel.
5. Staining the gel: After the electrophoretic run is completed, the separation patterns of the protein samples are visualized by staining the gels. This is done by exposing the gels to specific dyes which bind to proteins embed in the gels and help visualization of maximum number of protein spots. Commonly used dyes are Coomassie brilliant blue, silver stain and Sypro Ruby stain among others. Selection of the appropriate dye depends on the overall objective of the experiment. For example, if identity of the protein spots needs to be established by MS analysis then Coomassie brilliant blue stain is preferred over silver stain despite the sensitivity of the latter being higher by ten-fold. Coomassie dye interacts with the proteins embedded in the gel by non-covalent forces like electrostatic and Van der Waals interactions. Dye that is not bound to the protein diffuses out of the gel during destaining step. The proteins then appear as blue spots or bands on a clear background. Gels are then subjected to a corresponding destaining step before they are scanned using a gel documentation instrument.
6. Scanning of the gel: The gel documentation instrument usually consists of a chamber and a detector which can capture an image of the stained gel. The gel is place on the imaging platform, taking care that it does not break during the transfer. An image of the gel is captured and stored with an appropriate label. A representative image is shown in Fig.3. Such images of the stained gels 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 run n SDS-PAGE and stained with Coomassie blue.
7. Densitometric analysis: Apart from molecular weight determination, SDS PAGE 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. Here we will discuss the different steps associated with densitometric analysis of SDS-PAGE using Image Quant TL (IQTL) software (GE healthcare).
1. Open the software window.
Fig 4. Different options in analysis software
2. Click on the “1D gel analysis”.
3. Import your gel image and perform needful cropping and alignment of the image.
Fig 5.Selection and uploading of gel image.
4. Adjust image contrast to visualize the protein bands in each lane properly.
Fig 6. Adjusting image contrast and brightness
5. To create lanes automatically or manually select the appropriate button in the Navigator.
Fig 7. Creation of lanes in the gel image
6. If required, edit the lanes; resize/bend the lanes as per the band positions. Lanes can be moved and addition and deletion of lanes is also possible.
7. Perform background subtraction from all the lanes. Multiple background subtraction processes are available. Select one of those. Select “none” if you do not want to use background correction.
Fig 8. Background subtraction from all the lanes
8. To detect the band in every lane click the “Detect” button.The Software detects these bands automatically.
Fig 9. Detection of protein bands
9. Bands and edges can be edited either on the image or lane profile window. Right-click/left click to remove/add a band, respectively.
10. Use normalization mode to normalize the values against one or several known band. Select either their average volume or collective volume.
11. Export the data as excel files and perform subsequent analysis to interpret the data.
Fig 10. Export raw intensity values as excel format for further analysis