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Experiment-1: Gel-based proteomics to analyze human serum proteome
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Experiment-1: Gel-based Proteomics to Analyze Human Serum Proteome
Objective
To compare the serum protein expression profiles of diseased and healthy (control) human subjects using 2D gel electrophoresis.
 
THEORY
Proteins are complex bio-molecules with wide range of physiological functions in all living organisms. Various techniques have been developed over the last few decades by many researchers to study the structure and functions of proteins. Amongst all of the commonly used techniques, electrophoresis has been referred to as ‘Gold Standard’ due to its ability to provide much more information about protein properties. Two-dimensional gel electrophoresis (2DE) is one of the most widely used electrophoretic techniques for separation of complex mixtures of proteins. In 2DE, protein separation is carried out based on two different properties of proteins namely, isoelectric point and molecular weight. With the passage of time several advancements have been introduced in the separation procedure to increase the sensitivity, robustness and efficiency.
                                                         
1. Properties of proteins and separation
 
Proteins are biologically vital, complex biomolecules that possess a very wide range of functions compared to the other biomolecules. They possess dynamic physical and chemical properties which are largely dependent on their composition and structure. Unlike DNA, they do not possess uniform negative charge, which makes a very challenging task for studying these molecules using regular, conventional biochemical techniques. Proteins are long, polymeric, macromolecules made up of amino acid residues. Amino acids are molecules containing an amino group, a carboxylic acid group and an aliphatic or aromatic side chain that varies between different amino acids (Figure 1). 

 

 

 

 Figure 1. General structure of amino acid: An amino group, carboxylic group and R-group side chain.
 
The amino acid side chain, referred to as the R group, varies across the different amino acids, and thereby resulting in differences in properties for the various amino acids. The presence of acidic carboxylic acid group and basic amino group confers amino acids with an amphoteric nature i.e. the ability to behave as acids or bases. This results in amino acids bearing a different net charge depending upon pH of the surrounding environment. This property is utilized as one of the separation criteria for protein mixtures. Proteins have multiple structural levels; the primary structure consists of the linear arrangement of amino acids that are joined together by means of peptide bonds. The polypeptide backbone of the protein undergoes folding due to the formation of several internal bonds which gives rise to the secondary structure. Further interactions between the side chains of the amino acid residues and interactions between multiple polypeptide chains (subunit) lead to the tertiary and quaternary structures respectively.

 

 


 

Figure 2. Levels of structural complexity of proteins, the amino acid residues forming a helical structure to form a secondary state, connection between each polypeptide to form tertiary and quaternary structures respectively.
 
2. History and Development from electrophoresis 
Electrophoresis was invented by Prof. Arne Wilhelm Kaurin Tiselius; a Swedish scientist in 1930, for which he received the Nobel Prize in chemistry in the year of 1948. The concept of electrophoresis initially developed by him involved free solution and was termed as “moving boundary electrophoresis”. Reports of using filter papers and gels in electrophoresis were first seen only in early 1950 and rapid developments ensured electrophoresis to become a widely adopted technique by 1960 and later on.
 
 2.1 Advantages of 2DE over 1DE 
Initially proteins were separated by one-dimensional electrophoresis using molecular size as the separation criteria with smaller proteins migrating further along the gel than the larger ones. However, separation on the basis of only MW of the proteins was unable to provide comprehensive resolution of complex biological samples like serum, cell lysates, plant tissues etc. due to the complexity of the samples and presence of several protein molecules with similar or nearly similar molecular masses. To this end, in early 1956, Smithies and Poulik recognized a new approach in which a combination of two electrophoretic processes was carried out on gels at right angles to each other for better separation than single dimension run. The concept of 2DE was developed and established by O’Farrell in the year of 1975, which became rapidly accepted and adopted by many researchers as an extremely useful technique to study complex mixtures of proteins. As the name suggests, proteins are separated in 2DE based on two different properties - isolectric point (pI) in the first dimension and molecular weight (MW) in second dimension giving better resolution of hundreds to thousands of proteins in a single gel. In the year of 1977, 2DE approach was successfully employed for the first time by Anderson and Anderson for the analysis of human plasma proteins.   
 
3. Separation across two dimensions:
 
3.1 First dimension: 
Each protein has a specific isoelectric point (pI), at which it gains net neutral charge. This property is used for separation of proteins on the first dimension of 2DE. During IEF, the proteins with different charges move across the pH gradient strip due to the applied electric field. They come to rest at a position where their net charge is zero and they can no longer migrate in the electric field. Initially tube gels were used for first dimension separation in which a pH gradient was established using a suitable ampholyte solution consisting of low molecular weight organic acids and bases components.  These pH gradients were not very stable having tendency to breakdown upon application of concentrated samples thereby leading to a lot of variations and very less reproducibility in the results. The issues were resolved by replacing the tube gels with immobilized pH gradient (IPG) strips. IPG strips make use of immobulins that are co-polymerized with the gel and therefore eliminate the errors related to IEF separation. A wide range pH gradients on polyacrylamide gels are available commercially that can be selected depending on the complexity of sample to be used in the experiment. This innovative concept of IPG strips was developed by a German scientist, Angelika Görg. (Figure 3).

 

 


 

 

Figure 3. IPGphor unit used for Iso-electric focusing of the strip rehydrated with the sample.


3.2 Second dimension: 
The second dimension separation in 2DE involves SDS-PAGE, a commonly employed technique that separates molecules based on their molecular weights. Sodium dodecyl sulfate (SDS) is an anionic detergent that denatures the proteins and confers them with a uniform negative charge proportional to their molecular weight. This ensures that the charge-to-mass ratio of all proteins is nearly identical thereby facilitating separation completely on the basis of their molecular weights. The polyacrylamide gel is prepared using acrylamide and bisacrylamide as the cross-linkers for the polymerization reaction, which is aided by ammonium persulfate (APS), while N,N,N',N'-Tetramethylethylenediamine (TEMED) acts as a free radical supplier to facilitate the reaction in presence of tris-HCl and SDS solution. The gels casted forms the base for loading the focused IPG strips used in the 1st dimension, the whole gel setup is placed in the electrophoresis unit containing running buffer solution. Application of an electric field results migration of the proteins through the gel based entirely on their molecular weights. Once electrophoretic separation get completed, the proteins are stained with routinely used staining agents like coomassie blue and silver that allows the visualization of the separated protein spots. More recently, different fluorescent stains such as SYPRO Ruby Red, SYPRO Orange, Deep Purple, CyDye etc. have been developed, which allow for extremely sensitive detection of even very minute amounts of proteins.

 

 

 

Figure 4. Action of SDS on proteins during 2D run. Treatment with SDS denatures the proteins; further SDS adds negative charges all along the length of protein.

2DE has the ability to separate around 2000 proteins simultaneously in a single run, which makes it a highly effective and versatile tool to study relative protein concentrations in complex protein mixtures (Figure 5). This gel-based proteomic approach serves as a separation technique for resolving large numbers of proteins, and acts as an effective profiling tool to monitor relative abundances of the proteins through appropriate staining procedures. 2DE is widely applied to study proteome profiles that portray alterations in protein expression level, isoforms or post-translational modifications.

 

 

 

Figure 5: Pictorial representation of 2D electrophoresis experiment.
 
However, comparative analysis of multiple samples using 2DE found to be challenging, since only one sample could be resolved per gel. Gel-to-gel variations, manual artifacts, system variations, and lack of reproducibility made it difficult to compare gel patterns when samples (test and control) are resolved on two different gels. The requirement for a stable and robust method which could overcome this hurdle led to the introduction of difference gel electrophoresis (DIGE), developed by Mustafa Unlu and his group in1997This technique allows the separation and detection of up to three different protein mixtures on a single 2DE gel. It enabled researchers to study the relative expression of samples with respect to varied biological conditions such as disease, treated, post/pre treatment with high reproducibility. 2D-DIGE uses fluorescent cyanine dyes, Cy2, Cy3 and Cy5 (wavelength of ~488, ~532 and ~642 respectively), each having a specific excitation energy and wavelength that enables the accurate detection of the proteins labeled with each dye. Two different samples (test and control) are labeled with either of Cy3 and Cy5, while a pool of samples in equal quantity known as “internal standard” is labeled with Cy2. All the labeled samples are mixed together to run on a single gel. The internal standard is used to overcome gel-to-gel, manual, system and experimental variations. Application of the pooled internal standard having all the proteins from each experimental group minimizes any variability that may arise in the gel, thereby providing an efficient and accurate technique for protein profiling. Once separation of proteins takes place across the two different dimensions, the gel is scanned and visualized at different wavelengths allowing the comparative analysis of the relative expression patterns based on the spot intensity. The gel images are analyzed using advanced software which accurately quantifies the differential expression of many proteins between test and control samples on a single gel.

 

 

 

Figure 6. Pictorial depiction of a typical 2D-DIGE experiment, two different samples for the study are labeled with either of Cy3 and Cy5 and pool of these two samples in equal quantity is labeled with Cy2. Once the labeling reaction is completed, all the three labeled samples are pooled together to carry out 1st and 2nd dimensions separation of the labeled proteins. After electrophoretic separation 2D-DIGE gels are scanned employing suitable excitation/emission wavelengths for each of the CyDye [Cy3 (523/580 nm), Cy5 (633/670 nm) and Cy2 (488/520 nm)].
 
 
Human serum proteome
Serum is a complex biological fluid containing few highly abundant proteins and many low abundance proteins. Studies have shown that there are around 20 high abundance proteins in serum, which usually decrease the resolution and tend to mask the presence of other low abundance proteins. Albumin and immunoglobulin G (IgG) account for a very large fraction of the overall protein contents of serum. In order to obtain effective separation on 2DE, it is ideal to remove these highly abundant proteins from serum prior to electrophoresis process.
 
Table 1: List of 20 highly abundant proteins present in human serum

 

Albumin Apolipoprotein A-II Complement C1q Ceruloplasmin
IgG

Apolipoprotein A-I

Complement C4

a2-Macroglobin
IgA

Apolipoprotein B

Complement C3

Haptoglobin
IgM Transferrin a1-Acid Glycoprotein Prealbumin
IgD Fibrinogen

a1-Antitrypsin

Plasminogen

 


 

Removal of these high abundance proteins is usually carried out with the help of commercially available kits, which work on the basic principle of affinity chromatography. The columns present in the commercial kits are packed with affinity beads that are capable of binding specifically to high abundance proteins. High abundance proteins, which need to be removed, bind to the surface of the beads, while proteins of interest (other than albumin and IgG) elute out of the column by changing the pH of the buffer. Serum depletion can also be performed using size exclusion criterion wherein molecular cut-off columns are used. These columns retain the proteins that are above its cut-off range but release the lower molecular weight proteins. For example, a molecular cut-off column of 60 kDa will trap albumin having a molecular weight of 66 kDa.
The whole procedure of 2-DE can be subdivided into the following 4 steps, each of which will be described in detail.
1)      Sample Collection and Preparation
2)      First dimension - Isoelectric focusing
3)      Second dimension - Separation on SDS-PAGE
4)      Gel staining and data analysis
 
1) Sample Collection and Preparation  
  1.A - Sample Collection
Blood is composed of different types of blood cells and plasma. The plasma acts as a suspension liquid for blood cells and other biological molecules. Plasma that is deprived of coagulation factors is referred to as serum and contains all circulatory proteins except fibrinogen and other clotting factors. The collection of blood and separation of serum for proteomics experiments is the first crucial step that influences the result and reproducibility of the experiment. Proper care during sample handling prevents the denaturing of proteins in serum.
 
Sample collection is carried out as described below.
  •  Around 5 ml of intravenous blood is collected into a commercially available blood collection tube, which does not contain any external anti-coagulating agent. The Vacutainer tubes are commonly used to bring about efficient and rapid withdrawal of blood.
  • Immediately after collection, the tube is placed on ice for 30 min. This incubation time is required to bring about coagulation of the blood and formation of the fibrin clot, which is essential to separate the serum.
  • The serum is then separated by centrifuging the coagulated blood sample at 2500 rpm at 20°C for 10 min. The blood clot containing the different types of blood cells and clotting factors forms the pellet while the serum forms the supernatant.
  • The dark, yellowish, viscous supernatant is then carefully aspirated out using a micropipette and collected into a fresh, clean, labeled microcentrifuge tube. Care should be taken not to disturb the pellet.
  • Usually, about 1.5-2 ml of serum can be collected per 5 ml of whole blood.
  • The tube containing the serum is then labeled appropriately and stored at -80°C until further use.
 
 1.B- Sonication of serum proteins
 Biological fluids like serum are extremely complex and have a wide variety of proteins, which can hinder the separation in 2-DE. In order to improve the quality of protein extraction, various physical methods and chemical methods have been developed which reduce the complexity of the crude serum sample. Sonication of serum protein is one of the widely used physical methods to distort cell complex and its inter- and intra- protein interactions.  In this process, high frequency sound waves are applied to the crude samples for a short exposure,which help in disrupting the cells. Procedure for sonication of the crude serum sample is as follows:
 
  •  A fresh, clean, autoclaved microcentrifuge tube is taken and 200 µL of serum is pipette into it.
  • The serum is then diluted five times using Phosphate buffer, pH 7.4, after which the contents are mixed thoroughly by vortex for 30 sec.
  • The sample is then sonicated for 6 cycles of 5 sec pulse with a 59 sec gap in between each cycle at 20% amplitude.
  • Since the process of sonication generates a large amount of heat, one must ensure that the sample tube is always kept immersed in an ice bucket to avoid any denaturation or spoiling of the sample.  
  • Do apply ear plugs while doing the experiment, since the sounds bursts may cause some serious damage to ears.

 

 

Fig 8: Sonication of serum protein sample, required quantity of buffer is added to the sample. The probe is made to submerge into the sample solution; the tube is placed on the ice and sound waves are exposed for short time to carry out sonication.
 
1.C- Protein Precipitation using Acetone
In this process, acetone is used to precipitate out proteins from the solution, while ethanol is used as a washing agent. Addition of ice-cold acetone to the sample brings about precipitation of proteins from the solution, while the lipids, impurities and any detergents that may be present remain in the supernatant. The precipitated proteins are then harvested by centrifugation and the pellet containing desired proteins is washed with ethanol to remove any organic contaminants that may be present in the sample. (Note: Acetone to be used for precipitation must be kept for at least 5 h at -20°C before use).
 
The pellet obtained after centrifugation is then rehydrated using rehydration solution having the following composition:
 
  • 8 mM Urea: Urea, being a chaotropic agent, helps in stabilization and unfolding of proteins so that the protein’s charged surfaces are exposed towards the solution.
  •  2% w/v CHAPS {3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate}: This is a zwitterionic detergent used for solubilizing proteins.
  • 40 mM DTT (Dithiothreitol): DTT efficiently reduces inter and intra molecular disulphide interactions thereby exposing the ionic surfaces of the proteins.
  • 12 µL TBP (Tributylphosphine): TBP is a reducing agent which increases solubility of the protein. Due to its non-ionic nature, TBP does not migrate in the Immobilized pH gradient (IPG) strip during first dimension separation and therefore helps in maintaining reducing conditions.
  •  IPG buffer: This buffer is added in accordance with the IPG strip that is to be used for first dimension separation.
  •  All these chemicals are mixed to prepare the rehydration solution, aliquots of which is prepared and stored at -20°C for future use.
 
The detailed procedure for protein precipitation using acetone is as follows:
  • 1 ml of pre-processed serum is first divided into 5 parts, each containing 200 µL. Each sample is then mixed with 4 times the volume of acetone (800 µL). This turns the solution turbid, indicating the precipitation of proteins. The tubes are vortexed for about 15 sec to ensure proper mixing.
  • These tubes are then stored at -20°C for 6 h for incubation.
  • After this, the tubes are centrifuged at 1000 g for 15 min at 40°C. This helps in complete precipitation of proteins and formation of pellet.

 

Fig.9: TCA-Acetone precipitated proteins showing the white precipitation pattern. The white clouds determine the proteins getting precipitated to get to the bottom.
 
  • Carefully discard the supernatant, and add about 200 µL of ethanol for washing the pellet. Vortex the tube for 20 sec.
  • Centrifuge the tube at 5000 g for 10 min at 40°C. This separates the supernatant and forms a pellet of protein. The supernatant is discarded and the same step is repeated once more.
  • The pellet obtained is allowed to dry for about 15 min at room temperature. Since acetone is a highly volatile the excess of it evaporates leaving the pellet dry.
  • Rehydrate dry pellet using 100 µL rehydration solution. The tubes are vortexed to ensure proper re-suspension. If required about 20µL more of rehydration solution can be added to re-dissolve all the suspended particles.
  • Finally all the re-dissolved fractions of respective samples are pooled to make a single aliquot. This can be readily used for further work or can be stored in -20°C.
           
1.D: Removal of high abundant proteins:
Commercially available serum depletion kits contain affinity chromatography based columns along with relevant buffer solutions and collection tubes. The procedure for removal of high abundance proteins involves 2 major steps - column preparation and protein binding with elution.
 
(a) Column preparation: Commercially available kits are provided along with chromatography bead slurry, which is either given separately such that the column must be carefully packed by the user; or in the form of a packed, ready-to-use column.
 
  • The column needs to be activated to bring in the proper charged surfaces to facilitate the binding of desired proteins. This is done by passing the column with stipulated amount of activation buffer provided with the kit.
  • Once the column is applied with the activation buffer and fixed with collection tube it is centrifuged with recommended time and RPM.
  • The collected wash is discarded and the procedure is repeated.
  • Care should be taken to ensure proper activation of the affinity beads with the buffer resulting in proper binding of the proteins.
 
b) Protein binding and Elution: In this step, the desired proteins are bound on the column matrix by spinning the column with the protein sample. The chemistry of the column is such that the abundant proteins bind to the surface of beads containing specific affinity agents that capture the proteins. The unbound proteins (proteins of our interest), on the other hand, elute out of the column. Therefore it is very crucial step to collect the unbound proteins in a fresh, clean tube and store them under proper conditions for further use.
 
  • The column is first loaded with the recommended amount of protein sample, after which it is incubated for specified time duration in order to facilitate proper binding.
  • The column in then centrifuged with a fresh collection tube at the outlet and the collected flow-through is stored and labeled as flow through (F1).This fraction contains proteins of our interest i.e serum deprived of high abundance proteins.
  •  Next, the column is washed with the provided wash buffer by passing a specified volume of buffer into the column. It is centrifuged to collect the flow-through (F2) in a separate tube. This fraction contains loosely bound proteins, which were not eluted out on the first attempt.
  • The fractions, F2 and F1, can then be mixed to make a whole fraction of serum containing most of the low-abundance proteins.
 
 
1. E: Desalting: 
Serum not only contains proteins but also constitute other components such as nucleic acids, lipids, detergents, salts from buffers used etc. These can hinder the overall process of protein separation in the first dimension, which depends on charge of the proteins, and also in the second dimension by forming streaks in the gel image. Therefore, the sample should ideally be depleted of any other charged components in order to obtain better separation of spots.
 
This can also be done with the help of commercially available kits. The principle involved is similar to that of acetone or TCA precipitation of proteins.
  • The precipitating agent in the kit is added to the protein solution, which selectively precipitates out the proteins and leaves other unwanted ionic impurities in solution.
  • The precipitated proteins can be separated from the supernatant with the help of centrifugation.
  • The protein pellet remaining after pipetting out the supernatant is then re-suspended in a rehydration buffer.
 
 
 
 
 1. F: Quantitation: 
Quantification of proteins in a sample is a crucial step for 2-DE since every IPG strip has an specified optimal protein intake capacity. It is also important to load similar protein quantities on different gels so that comparison across gels is feasible. This also helps in avoiding experimental artifacts and allows analysis of the gel in a biological context. The protein estimation method should be selected in such a way that the reagents used for the method must be compatible with other added chemicals and must not interfere in the 1D and 2D separations.
The modified Bradford method is conventionally used by many researchers due to the ready availability of reagents in the market and ease of use. The Bradford reagent contains Coomassie Brillient blue G-250 dye, which is initially brownish red in color and eventually changes to a blue color with protein binding, which can be read at 595nm.
Note: The sample concentration will depend on the range of the standards used. If the sample concentration is above the limit of reading of colorimeter, then the sample must be diluted and assay must be repeated. Finally, the result must be multiplied by the dilution factor to obtain the original undiluted protein concentration. If, on the other hand, the concentration is below the lowest standard, reduce the standard concentration to carry out the assay.
 
 
2. First dimension - Isoelectric focusing
 Isoelectric focusing constitutes the first dimension separation in 2-D electrophoresis. This technique separates proteins on the basis of their respective isoelectric points (pI) i.e the pH at which the net charge on the protein is zero. Focusing is carried out with the help of commercially available Immobilized pH Gradient (IPG) strips on which, the proteins move under the influence of an electric field until their net charge is zero and get focused at their particular pI.
 
IPG strips are solid surfaces on which dehydrated polyacrylamide is coated. The different derivatives of acrylamide having a range of pKa values are found across the gel, which enables separation of proteins in the first dimension. These commercial strips are available in a range of pH gradients such as 4-7, 3-10, 4-11 etc. as well as, in a variety of lengths starting from 7, 11, 13, 18 and 24cm. The user can choose the strip based on the protein separation across the length and pH requirements in a particular experiment. The strips are rehydrated either with only rehydration buffer prior to protein loading or incase suitable protein concentration along with rehydration buffer can be used. The IPG strips are then soaked with rehydration solution for overnight as in for passive rehydration or by applying current for active rehydration. The focusing is carried out on an instrument that enables gradient supply of electric current to the IPG strips.  Once the proteins have been resolved in the first dimension, the strips are loaded on the SDS-PAGE gel to proceed with second dimension separation.
 
The detailed procedure for isoelectric focusing can be subdivided into following categories:
 
2.A. Rehydration of IPG strips:
Commercially available IPG strips contain the polyacrylamide gel in its dehydrated form. The strips therefore need to be rehydrated before they can be loaded with the protein sample. This is done by placing the strip in the rehydration tray containing the rehydration solution, overnight, for about 10-12 h. This enables efficient absorption of proteins on the gel. The procedure for strip rehydration is as follows:

  • The rehydration solution containing 8 M urea and 4% CHAPS/TRITON X100 is prepared. The components are mixed well and stored at -20°C.
  • 5 µl of the required IPG buffer and 6.2 mg of DTT are added just prior to use to 1 mL of the rehydration solution and the components mixed thoroughly by vortex.
  • The rehydration tray is cleaned thoroughly, and the dry lanes are loaded with the rehydration buffer.
  • The IPG strip to be rehydrated is selected and the protective cover that is present on it is removed carefully using forceps.
  • Each IPG strip has two sides; a base which is made of plastic and the other side having the gel on it.
  • The IPG strip is then carefully placed into the lane using forceps such that the surface with gel faces downwards and gets maximum contact with the solution around it.
  • The set-up is then left undisturbed for about 20-30 min.
  •  Mineral oil is applied on the set-up to avoid the drying of gels. It is advisable to apply mineral oil to two adjacent lanes so as to supplement uniformity of oil.
  • The set-up is left on the working bench for 15 hours.
  • The manifold of the IEF instrument is then cleaned and placed on an even surface to prepare for focusing.
  • The IPG strips are removed from the rehydration tray with the help of forceps and the ends are gently dabbed over an absorbent tissue to remove any excess mineral oil.
  • The strip is then carefully placed in the fresh tray, with the gel side facing upwards. Wicks are then placed on each end of the gel and other wells are filled with fresh mineral oil.
  • Electrodes are carefully placed over the assembly and the desired programme with suitable voltage gradient over time intervals is set to start the focusing.
  • The voltage gradients and time intervals are decided based on the strip being used and the sample being run. Optimization of these critical parameters is required to get best resolution in the first dimension. The programme can end with a holding step which works on a low voltage for stipulated amount of time.
  • The separation can be monitored real-time. The wick in use may need to be changed in middle of experiment if its color changes to yellow. This coloring of the wick takes place because of the excess salts and impurities that may be present in the sample.
  •  Once focusing is complete, the strips are taken out of the tray and the ends gently dabbed on the surface of an absorbent tissue to remove excess oil.

The focused strips are now ready for the next step

 

 


 

 
Fig.10: Schematic representation of isoelectric focusing of proteins starts with the rehydration of strip with sample followed with placing the strip on the manifold, pouring the mineral oil in the lanes to carry out focusing.
 

 

 
Fig.11: Stepwise representation of Isoelectric focusing of proteins. a) Assembly the manifold on Instrument b) Rehydrated strip is placed in the lane c) Mineral oil is poured to prevent drying of strip d) Wicks are placed on either side e) Electrodes are placed across the strip f) The IEF lid is closed to start the focusing .


 

 

 

Fig.12: Representation of separation of proteins on an IPG strip, the tracking dye moves faster than protein indicating the progress of separation step.
 
2. B Equilibration of strips:
The isoelectrically focused strips must be equilibrated before they are separated in their second dimension. This step ensures proper denaturation of proteins, which in turn enables efficient separation based on molecular weight during SDS-PAGE. Dithiothreitol (DTT) is used for reduction of inter and intra-chain disulfide bonds in proteins. Iodoacetamide, (IAA) being a potent alkylating agent, prevents the reformation of these broken disulfide bonds by alkylating the sulphur atoms.
 
The equilibration solution consisting of 6 M urea, 75 mM tris HCl buffer, 29.3% glycerol, 2% SDS and 0.02% bromophenol blue, made up to 20 mL using distilled water, is prepared. The contents are thoroughly mixed and can be stored at -20°C for later use. Just before use, 10 mg of DTT is added to 10 mL of the equilibration solution to obtain solution X and 25 mg of iodoacetamide is added to the remaining 10 mL of the equilibration solution to prepare solution Y.
 
First, solution X is taken in a container and the focussed strip is placed for around 10 min treatment. Next, the strip is transferred to solution Y where it remains for another 10 min, thereby completing the last step of focusing. The strip is then ready for separation in the second dimension.

 

 

 

Fig.13: Equilibration of proteins on IPG strips before transfer to second dimension. The focused strip is treated with equilibration buffer 1 for 15 min and transferred to equilibration buffer 2 for another 15 min, finally on to the SDS-PAGE gel, sealed with agarose to prepare for the run.
 
3. Second dimension - Separation on SDS-PAGE
SDS-PAGE, which separates proteins on the basis of their molecular weight, constitutes the second dimension of 2-DE after isoelectric focusing. The gels used for the 2D run must be casted and kept ready at least half an hour so that there is sufficient time for proper gel polymerization. The concentration and size of the gel vary based on the sample being separated and the dimensions of the IPG strip used during first dimension, separation. The commonly used and preferable gel concentration is 12.5%. The IPG strip containing the separated proteins, being very sleek and fragile, must be placed carefully and firmly in contact with the SDS-PAGE gel, such that there are no air gaps between the two surfaces that could allow the protein to escape. To facilitate this process, an agarose solution is often used as a fixing agent, which helps in removing any air bubbles that may be formed. Bromophenol blue (BPB), which is added to the agarose solution, acts as a tracking dye for the experiment to view the electrophoretic migration at any given time. The detailed protocol is as follows:
 
First, the appropriately sized gel casting apparatus is assembled together.
Next, the following components are prepared and mixed together for casting the gel (indicated contents are optimum for casting an 11 cm gel):
Acrylamide bis-Acrylamide solution (29:1 ratio) -3.14 mL
Distilled water-3.685 mL
Tris-HCl (pH 8.8)-2.435 mL
SDS (10%)-93.75 µL
APS (10%)-93.75 µL
TEMED-3.75 µL
A 10X stock solution of the buffers required for the gel tanks are prepared by mixing the following:
Tris base (FW 121.1) 250 mM—30.3 g
Glycine (FW 75.07) 192 mM—144.1 g
SDS (FW 288.38) 0.1% (w/v)--10 g
Double-distilled water to--1 L
This 10X stock solution can be diluted to 1X strength before use (100 ml made up to 1000ml).
 
Acrylamide Bis-Acrylamide solution together form polymer and form the bases of the gel. SDS helps for providing uniform negative charge on the protein surface which helps for movement proteins in PAGE depending on molecular weight regardless of charge. APS along with TEMED will form free radicals which help in polymerization of gels. Note that the TEMED can be added just before pouring the gel.
  • Add all the contents mentioned above. Mix it thoroughly.
  • Slowly pour the gel through the sides of the plates by making contact with the sides to avoid the formation of air bubble.
  • Keep in mind that a small space has to be left for placing the IPG strip and pouring agarose solution on the top.
  • Once the gel is poured immediately pour a small quantity of water with 0.1% SDS on top. This maintains a uniform surface for placing the IPG strip.
  • Gel takes approximately 35-40 min to polymerize.
  • Mean while prepare 0.75% agarose solution with 0.02% of BPB in water. Mix the content and warm to dissolve the content. After dissolution cool it a bit so that it is optimum to pour.
  • Decant the water on gel carefully remove the remaining water with the help of absorbent tissue paper.
  • Carefully take equilibrated IPG strip, place on the gel so that the strip ensure proper contact of strip with gel
  • Carefully pour the agarose solution till the top mark and allow it to solidify.
  • Place the set-up inside the tank of electrophoresis and pour the tank buffer into inner and outer tank.
  •  Apply constant voltage of 90 volts. The procedure approximately takes 2-3 h for separation.
  • The electric supply can be stopped when the dye front reaches the end of the gel or slightly moves out of the gel.

 

 

 

 

Fig.14: The set unit for SDS-PAGE starts with a) Gel casting assembly b) Casting of gel c) placing the focused strip with sealing with agarose d) Setting up for separation by pouring tank buffer e) Typical experimental set-up unit for SDS-PAGE.
 
 
4)  Gel staining and data analysis
     
 4. A: Staining and destaining 
Once the proteins are separated on SDS PAGE, to visualize the separation patterns, proteins need to be stained. Stains are chemical components which specifically bind to proteins enabling the visualization. The property of dye should be such that it helps visualization of maximum number of protein spots on gel with high amount of sensitivity.
 
 
Coomassie brilliant blue is most widely used stain. There are some more varieties such as silver staining, sypro ruby etc. Depending up on sensitivity and cost effectiveness the selection can be done unlike stains of silver do interfere at protein identification during MS The sensitivity of silver is ten-fold more as compared to coomassie in visualizing proteins at lower concentration. So selection of the dye, depending upon the requirement is a user call.
 
Image of stained gel can be taken using gel documentation instrument which will typically have a chamber and a detector for capturing image. Comparison of the global expression profiling or differential expression of proteins within or across the gel can be done with the help of commercially available software.
 
Staining procedures are as follows; Preparation of coomassie dye;
Methanol, acetic acid and distilled water are mixed in 40:7:53 ratios. To same 350mg Coomassie Blue is added. The solution is mixed well and can be filtered using a filter paper to get rid of un-dissolved debris. Though the stain is mostly used once, it can be used thrice efficiently. The gel after removing and before staining has to be washed with distilled water. This helps the removal of SDS which may hinder the proper staining of the gel by forming streaks all over the gels.
 
  • Carefully remove the gel out of the gel plates.
  • Place the gel into container with distilled water; wash the gels briefly at least thrice by changing water. Then discard the water used.
  • Now immerse the gel in stain solution, cover the container, and keep it on gel rocker with a constant shacking for about 6-8 h.
  • The stain covers the whole gel including the parts without proteins. Hence the gels have to be distained so that only the part with proteins will retain the stain and rest of the background is clear.
  • Take the gel out of stain and immerse in distaining solution (40:7:53 methanol, acetic acid, water) for about 5-6 h. Change the stain about three times for efficient destining.
  • The stained gel can be washed with distilled water twice before imaging.

 

 

 

Fig.15: Image of a typical 2-DE gel showing serum proteins separated on a 4-7 pH range on the X axis and molecular weight on the Y axis
 
 4. B: Imaging and analysis
 
Once the proteins are separated over 2DE, each spot on gel represents unique protein/proteins specific to particular pI and molecular weight. Perhaps protein spots on the gel can be interpreted in various ways depending upon the user’s application. If the user wishes to have application such as global protein expression profiling or differential expression profiling it is tough to do so manually for there are enormous numbers of protein spots over a gel and manual interpretation may lead into an erroneous result. Hence the gels can be analyzed with the aid of commercially available software, which will enable user to compare the expression profiles of proteins within and across the gels. Software uses unique way of reading pixel intensities for implying the quantity of the proteins. Analysis gives a comprehensive output in statistical terms which are often easy to interpret and can be extended to a wider biological scenario.
 
The gel is placed carefully on the imaging platform. Care should be taken to ensure that the gel does not break while transferring.The gel image is captured and saved with appropriate label. Image analysis is performed for checking the differential expression between two different samples with the help of commercially available software packages. These software’s have tools which allow gels to be cropped, overlayed, zoomed into, edited, later matched, compared with, derive statistical data for the analysis. Although there are several tools which need to be explored in detail with the software, the basic steps for image analysis is as follows:
 

  •  A suitable labeled master folder is first created which should contain the gels to be analyzed. The gel images to be analyzed are imported, opened on the software and labeled appropriately.  
  • The region on the gel having maximum spot density is selected and the gel is cropped with the cropping tool so that regions without spots are excluded from the analysis.
  • The spots on the gel can be selected with the help of the spot picking tool on the software. These spots are selected by the software on the basis of certain user-defined criteria, which gets translated by the software into pixel intensities of the spots.
  • The gel can be zoomed into and carefully analyzed to detect spots that may have been selected based on the defined criteria to retain actually protein spots.  This step is very important because the selected spots will be considered for comparison across the gels. Therefore great care must be taken not to select a wrong spot and more importantly, not to miss an important protein spot.
  • The selected spots are then matched across gels. This process displays the number of spots that are matched and unmatched across the gels.
  • The extent of matching can also be seen with a 3-D graphical representation of the spots.
  • The spot parameters such as volume, intensity, possible pI, molecular weight etc. can be obtained through the software.
  •  Statistical parameters of spot matching across different gels such as coefficient of variation, standard deviation, t-test values etc. can also be obtained.
  •  All this data can be compiled together to understand the differential expression of proteins across the desired gels.

 

 

 

 

Fig.16: Representation of analysis through commercially available software. The steps start with loading of the gel image, carrying out cropping, making the necessary settings for spot detection, comparing and checking the appearance of spots in 3D view for proper matching.


 

 

 

 

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