SDS PAGE, WESTERN BLOTTING, AND ELISA

This presentation includes the principle involved, chemistry, procedure, and application of various advance molecular biology like SDS PAGE, Western Blotting, and ELISA.
SDS PAGE is widely used to analyze the proteins in complex extracts.
The polyacrylamide gels are used to separate proteins.
Polyacrylamide is inert, and hence, shows no interaction with the protein being separated and forms a matrix.
Size of the pores in the gel can be controlled by adjusting the concentration of acrylamide.
Acrylamide undergoes polymerization in order to form a gel. Hence, APS (ammonium per sulphate) & TEMED (N,N,N’,N’-tetramethylethylenediamine) are added to initiate the process of polymerization.
It's application includes separation of protein mixture on separating gel and their identification using different techniques like western blotting.
Western blotting, also known as immunoblotting or protein blotting, is a core technique in cell and molecular biology. In most basic terms, it is used to detect the presence of a specific protein in a complex mixture extracted from cells.
Western blots are effective in detecting low nanogram to low picogram amounts of target protein, depending on the antibodies used and the detection substrate chosen. If the target is suspected to be of very low abundance, or if there is no detectible signal on the blot, then it may be necessary to concentrate, immunoprecipitate, or fractionate the starting material.
This technique is used to study cell signalling pathways, cell cycle pathways, drug action pathways, protein-protein interaction.
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying peptides, proteins, antibodies, and hormones.
In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme.
Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product.
The most crucial element of the detection strategy is a highly specific antibody-antigen interaction.
ELISA begins with a coating step, in which the first layer, consisting of a target antigen or antibody, is adsorbed onto a 96-well polystyrene plate.
This is followed by a blocking step in which all unbound sites are coated with a blocking agent.
Following a series of washes, the plate is incubated with enzyme-conjugated antibody.
Another series of washes removes all unbound antibody.
A substrate is then added, producing a calorimetric signal. Finally, the plate is read.
It's types include Direct ELISA, Indirect ELISA, Sandwich ELISA and competitive ELISA. This technique is used to determine serum antibody concentrations, potential food allergens (milk, peanuts, almonds), detection of antigens and antibodies, disease outbreaks.

1. Presented by Parul Sharma PGD20225872 M.Pharm Pharmacology 1 SDS PAGE, WESTERN BLOTTING, AND ELISA

2. SDS PAGE (Sodium dodecyl sulfate Polyacrylamide Gel Electrophoresis) SDS-PAGE is widely used to analyze the proteins in complex extracts. The most used methods are derived from the discontinuous SDS-PAGE system first described by Laemmli (1970). The system consists of two gels - a resolving (aka running) gel in which proteins are resolved based on their molecular weights (MWs) and a stacking gel in which proteins are concentrated prior to entering the resolving gel. Differences in the compositions of the stacking gel, resolving gel and electrophoresis buffer produce a system that is capable of finely resolving proteins according to their MWs. 2

3. Chemistry of acrylamide polymerization The polyacrylamide gels are used to separate proteins. Polyacrylamide is inert, and hence, shows no interaction with the protein being separated and forms a matrix. Size of the pores in the gel can be controlled by adjusting the concentration of acrylamide. Acrylamide undergoes polymerization in order to form a gel. Hence, APS (ammonium per sulphate) & TEMED (N,N,N’,N’- tetramethylethylenediamine ) are added to initiate the process of polymerization. 3

4. APS & TEMED are added to the very last step of gel preparation because the process of polymerization of acrylamide starts as soon as TEMED is added to the preparation. APS generates acrylamide free radicals so that free radical polymerization can be initiated. TEMED is a free radical stabilizer and is added to promote polymerization. 4

5. Proteins are denatured prior to electrophoresis Proteins show tremendous variation in their amino acid compositions and in the distribution of amino acids in their folded structures, features with important implications for electrophoresis. Because proteins are so diverse with respect to their surface charges and geometries, the molecular weights of folded proteins cannot be simply determined by their migration rate in an electric field. Positively and negatively charged proteins would migrate in different directions. 5

6. 6 To resolve the proteins in a sample according to their size, proteins are converted to a uniform geometry by binding to amino acids in protein and imparting a uniform charge/mass ratio to the proteins. In SDS-PAGE, the solution is to denature the proteins by boiling them with the anionic detergent, sodium dodecyl sulfate (SDS), and 2-mercaptoethanol. The combination of heat and detergent is sufficient to break the many noncovalent bonds that stabilize protein folds, and 2-mercaptoethanol breaks any covalent bonds between cysteine residues. SDS being an anionic surfactant when dissolved in water gives a net negative charge to the denatured protein. One molecule of SDS binds every 2 amino acids of the protein.

7. Discontinuities between the stacking and running gels underlie the resolving power of the SDS-PAGE gels The stacking and running (resolving) gels have different pore sizes, ionic strengths and pHs . Stacking gel contains less concentration of acrylamide and is prepared using Tris HCl buffer with pH 6.8 Resolving/separating gel contains higher concentration of acrylamide and is prepared using Tris HCl buffer with pH 8.8 SDS PAGE electrophoresis buffer is made using Tris glycine buffer with pH 8.3. Role of stacking gel is to ensure the movement of all loaded sample to be concentrated in one tight band before entering resolving gel. 7

8. Steps involved in SDS PAGE electrophoresis 8

9. 1. Gel Preparation Gel glass plates are prepared in the glass frame and placed in the casting frame. Comb is placed in between these glass plates. (mark approx. 1cm below the comb, the resolving gel is prepared up to this mark). To ensure that there is no leakage between the plates, we pour water in between. Components for gel preparation includes: Polyacrylamide Tris HCl buffer Sodium dodecyl sulfate (SDS) Distilled water APS & TEMED Prepared gel is then poured in between the glass plates, the comb is placed, and the gel is left undisturbed for solidification. 9

10. 2. Preparation of running buffer and sample Running buffer is prepared using Tris-glycine (pH – 8.3) Sample preparation includes the following components Protein mixture Tris HCl (pH – 6.8) SDS Beta- mercaptoethanol Bromophenol blue Glycerol Distilled water 10

11. Tris HCl buffer is used to maintain the pH SDS maintains the primary structure of protein and provides a negative charge to the protein. Beta mercaptoethanol is the reducing agent that breaks –SH bonds between proteins. Bromophenol blue dye is used to aid visualization. Glycerol provides viscosity to the sample so that it does not come out of wells. Distilled water is used for volume makeup. Running buffer is then poured in between the gel and the sample is loaded in the wells. The whole apparatus is closed and connected to an electrical circuit. 11

12. 3. Supply of electric current Once a voltage is applied, the chloride ions in the sample buffer and stacking gel move rapidly toward the positive pole, forming the leading edge of a moving ion front. Glycine molecules have a very little charge in the stacking gel, so they migrate at the rear of the moving ion front. This difference in chloride and glycine mobility sets up a steep voltage gradient in the stacking gel that sweeps along the negatively charged protein-SDS complexes. The large pores of the stacking gel present very little resistance to the movement of protein-SDS complexes, which then “stack up” into a very concentrated region at the interface between the running and stacking gels. Protein-SDS complexes remain concentrated at the interface until the slowly migrating glycine molecules reach the boundary between the two gels 12

13. Dramatic changes occur as the glycine ions enter the running gel. The pH of the running gel is closer to the pKa of the glycine amino groups, so a significant fraction of the glycine molecules assume a negative charge. Negatively charged glycine molecules begin to move at the same rate as the chloride ions, thereby eliminating the voltage difference that controlled protein mobility through the stacking gel. The pores in the running gel are much smaller than those of the stacking gel, so the pores present frictional resistance to the migration of proteins. Proteins begin to migrate at different rates, because of the sieving properties of the gel. Smaller protein-SDS complexes migrate more quickly than larger protein SDS complexes. Within a certain range determined by the porosity of the gel, the migration rate of a protein in the running gel is inversely proportional to the logarithm of its MW. Commonly used dyes for protein visualization are Coomassie blue and silver stain. 13

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15. WESTERN BLOTTING 15

16. Western blotting, also known as immunoblotting or protein blotting, is a core technique in cell and molecular biology. In most basic terms, it is used to detect the presence of a specific protein in a complex mixture extracted from cells. The Western blotting procedure relies upon three key elements to accomplish this task: the separation of protein mixtures by size using gel electrophoresis; the efficient transfer of separated proteins to a solid support; and the specific detection of a target protein by appropriately matched antibodies. Once detected, the target protein will be visualized as a band on a blotting membrane, X-ray film, or imaging system. 16

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18. Monoclonal and Polyclonal Antibodies 18

19. Genetically Engineered Antibodies In addition to traditional monoclonal and polyclonal antibodies targeted against specific proteins, there are other means of antibody generation and protein detection available as the result of numerous advances in genetic engineering technology. It is now possible to create and produce antibodies using fully in vitro techniques such as phage display in conjunction with highly complex libraries which represent the vast array of potential antibody binding regions 19

20. Epitope Tags 20 If there are no antibodies available to the protein of interest, it is still possible to carry out a range of immunodetection techniques, including Western blotting, by using epitope tags and matched epitope tag antibodies. This elegant strategy works by adding a small sequence of DNA that codes for a known antigenic epitope during the cloning of the protein of interest. Since matched antibodies already exist that will specifically bind to this epitope, the target protein can be detected because it also expresses the appropriate epitope. Therefore, immunodetection can be carried out quickly and without the need to wait for the generation of unique antibodies to a newly identified target protein.

21. A typical Western blot, or immunoblot, relies upon a purified, semi-purified, or crude extract of cellular proteins containing a target protein that can be detected by antibodies. Several key steps are required to take the sample from the cellular starting point to a detectible band on a Western blot. 21

22. The three key preparative stages are: • Sample production by lysis or homogenization to solubilize and release cellular proteins. • Separation of protein mixtures using gel electrophoresis. • Transfer of separated proteins to a blotting membrane which can be manipulated more easily than a gel 22

23. Western blots are effective in detecting low nanogram to low picogram amounts of target protein, depending on the antibodies used and the detection substrate chosen. If the target is suspected to be of very low abundance, or if there is no detectible signal on the blot, then it may be necessary to concentrate, immunoprecipitate , or fractionate the starting material 23

24. Sample Preparation Crude cellular lysates are the most common direct source of starting material used in Western blotting. They can be prepared from immortalized cell lines known to express the target protein, or from transfected cells carrying a protein expression vector. For best results, all these steps should be carried out in a cold room, or on ice. This will minimize proteolysis, dephosphorylation, and denaturation since all begin to occur once the cells are disrupted. Cell lysis takes place with the help of different detergents and lysis buffers are used (e.g. NP 40 lysis buffer, RIPA buffer, Protease inhibitors etc.) A blender is used to homogenize the tissue in PBS, and then cell lysis buffer is added. Once the tissue has been homogenized and lysed, the solubilized cellular components are clarified by centrifugation and tested for protein concentration prior to loading on a gel. 24

25. SDS PAGE protein separation After the samples have been prepared, they are separated by size using SDS-PAGE (sodium dodecyl sulpate -polyacrylamide gel electrophoresis). Since the samples have been denatured in a gel loading buffer containing SDS detergent, the protein is uniformly negatively charged and will now migrate in an electric field through the gel and towards the positive electrode. Since the charge-to-mass ratio is equalized by the binding of SDS consistently along the length of the proteins, and higher structure has been removed, the proteins will be separated primarily by size. The key is to effect a separation such that the target protein will be properly resolved from the other components of the mixture. This makes it possible to clearly identify the target protein later through immunodetection with a specific antibody. 25

26. Transfer of protein to membrane We need to transfer protein from the gel into a membrane. Usually, nitrocellulose or PVDF ( polyvinylidene difluoride) is used. Nitrocellulose has been in use for a long time and is sometimes preferred because of its excellent protein binding and retention capabilities. However, nitrocellulose is brittle and thus it is usually less effective when blots need to be reused. PVDF demonstrates superior mechanical strength making it suitable for stripping/ reprobing and for further protein characterization techniques, such as sequencing and proteolysis Why protein transfer?? Because in order to detect protein we are going to add probes. And probes can bind to different regions of the gel making alterations in our result interpretation. So the probe should attach only to the target protein. Another reason is that gel is very fragile because of its composition and hence rest of the blotting process of probing and reactions can not be done using gel. 26

27. 27 While it is possible to use diffusion or vacuum-assisted transfer, electroblotting ( Towbin et al.,1979) is the method relied upon in most laboratories, due to the speed and efficiency of transfer. Electrophoretic transfer can be accomplished under wet or semi-dry conditions. In a wet transfer, the gel/blotting paper/filter paper sandwich is placed into a cassette along with protective fiber pads. The cassette is then immersed in a buffer tank and subjected to an electrical field. With semi-dry transfer, the gel/blotting paper/filter paper sandwich is assembled on large electrode plates which generate the electric field, and buffer is confined to the stack of wet filter papers.

28. This prepared membrane is placed in a gel holder cassette, and this whole set is placed in a buffer tank with gel facing the cathode. Transfer buffer is generally composed of – Tris base (pH 8.3) Glycine Methanol Methanol removes SDS from protein and helps the protein to bond with the membrane to interact with antibodies. 28

29. Once the blotting step has been completed, the apparatus is carefully disassembled and the success of the transfer is evaluated. The simplest method of confirming the transfer involves noting the appearance of prestained markers on the blot as compared to the gel. However, this is only a crude measure since it provides results for a single lane. A more reliable method of confirming transfer is through the use of a reversible stain which identifies the presence of protein bands directly on the membrane 29

30. After blotting, the target protein will be detected using appropriately matched and labeled antibodies. The typical immunodetection stage involves a few basic steps: • Blocking - The blot containing the transferred protein bands is incubated with a protein or detergent solution which covers the entire surface so that antibodies do not bind non-specifically to the membrane. • Antibody incubation - Labeled antibody binds to the target protein band present on the blot in a one-step or two-step procedure. • Detection with substrate - The label attached to the antibody, usually an enzyme such as HRP (Horseradish Peroxidase), is detected using a substrate that produces a visible signal corresponding to the position of the target protein. 30

31. Blocking of nonspecific sites Blocking is a very important step in the immunodetection phase of Western blotting because it prevents the non-specific binding of antibodies to the blotting membrane. The most commonly used blocking solutions contain 3-5% BSA or non-fat dried milk (also known as Blotto or BLOTTO) in a solution of PBS (phosphate buffered saline) or TBS (tris buffered saline). Often, a small amount of Tween®20 detergent is added to blocking and washing solutions to reduce background staining, and the buffer is known as PBST or TBST. After blocking, the blot is rinsed in wash buffer. 31

32. Incubation of membrane with antibodies After blocking and washing, the blot will be incubated in a dilute solution of antibody, usually for a few hours at room temperature or overnight at 4°C. The antibody is diluted in wash buffer (PBST or TBST) or a diluted blocking solution, the choice depends upon the antibody. In the first step, Primary antibodies are incubated with the protein. Then a secondary antibody specific to the primary antibody is used with conjugated enzymes (HRP or alkaline phosphatase) or any other detecting compound. The secondary antibody shows no interaction with the protein itself. Washing is done to remove excess or unbound secondary antibodies. 32

33. Detection with Substrate The most common antibody label used in Western blots is HRP, a small, stable enzyme with high specificity and rapid turnover. HRP is deactivated by sodium azide , so it is imperative that no azide is present in the blocking, dilution, or washing solutions. The HRP label is detected when it is exposed to a substrate solution in the final step of the immunodetection procedure. Substrate solutions for Western blotting are chemical reagents that are acted upon by the enzyme to yield a signal that can be easily measured. HRP label is typically detected with either colorimetric or chemiluminescent substrates. 33

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35. What kind of studies can be done using western blot technique?? Study cell signalling pathways Cell cycle pathways Drug action pathways Protein-protein interaction 35

36. ELISA (enzyme-linked immunosorbent assay) ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying peptides, proteins, antibodies, and hormones. In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction. 36

37. ELISA begins with a coating step, in which the first layer, consisting of a target antigen or antibody, is adsorbed onto a 96-well polystyrene plate. This is followed by a blocking step in which all unbound sites are coated with a blocking agent. Following a series of washes, the plate is incubated with enzyme-conjugated antibody. Another series of washes removes all unbound antibody. A substrate is then added, producing a calorimetric signal. Finally, the plate is read. 37

38. ELISA Types 38

39. 1. Direct ELISA The direct detection method uses a primary antibody labeled with a reporter enzyme or a tag that reacts directly with the antigen. Direct detection can be performed with an antigen that is directly immobilized on the assay plate or with the capture assay format. Direct detection, while not widely used in ELISA, is quite common for immunohistochemical staining of tissues and cells. Advantages • Quick because only one antibody and fewer steps are used. • Cross-reactivity of secondary antibody is eliminated. Disadvantages • Immunoreactivity of the primary antibody might be adversely affected by labeling with enzymes or tags. • Labeling primary antibodies for each specific ELISA system is time-consuming and expensive. • No flexibility in the choice of primary antibody label from one experiment to another. • Minimal signal amplification. 39

40. 2. Indirect ELISA For indirect detection, the antigen coated to a multi-well plate is detected in two stages or layers. First, an unlabeled primary antibody, which is specific to the antigen, is applied. Next, an enzyme-labeled secondary antibody is bound to the first antibody. The secondary antibody is usually an anti-species antibody and is often polyclonal. Advantages • A wide variety of labeled secondary antibodies are available commercially. • Versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection. • Maximum immunoreactivity of the primary antibody is retained because it is not labeled. • Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. Disadvantages • Cross-reactivity might occur with the secondary antibody, resulting in a nonspecific signal. • An extra incubation step is required in the procedure. 40

41. 3. Sandwich ELISA Sandwich ELISAs typically require the use of matched antibody pairs, where each antibody is specific for a different, non-overlapping part (epitope) of the antigen molecule. A first antibody (known as capture antibody) is coated to the wells. The sample solution is then added to the well. A second antibody (known as detection antibody) follows this step in order to measure the concentration of the sample. This type of ELISA has the following advantages: • High specificity: the antigen/analyte is specifically captured and detected • Suitable for complex (or crude/impure) samples: the antigen does not require purification prior to measurement • Flexibility and sensitivity: both direct or indirect detection methods can be used 41

42. 4. Competitive ELISA The key event of competitive ELISA (also known as inhibition ELISA) is the process of competitive reaction between the sample antigen and antigen bound to the wells of a microtiter plate with the primary antibody. First, the primary antibody is incubated with the sample antigen, and the resulting antibody–antigen complexes are added to wells that have been coated with the same antigen. After an incubation period, any unbound antibody is washed off. The more antigen in the sample, the more primary antibody will be bound to the sample antigen. Therefore, there will be a smaller amount of primary antibody available to bind to the antigen coated on the well, resulting in a signal reduction. The main advantage of this type of ELISA arises from its high sensitivity to compositional differences in complex antigen mixtures, even when the specific detecting antibody is present in relatively small amounts. 42

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44. Applications of ELISA Serum Antibody Concentrations Detecting potential food allergens (milk, peanuts, walnuts, almonds and eggs) Disease outbreaks – tracking the spread of disease. E.g. HIV, bird flu, common colds, cholera, STD etc Detections of antigens E.g. pregnancy hormones, drug allergen, GMO, man cow disease Detection of antibodies in blood sample for past exposure to disease E.g. Lyme disease, trichinosis, HIV, bird flu. 44

45. Reference “ELISA Handbook Principle, Troubleshooting, Sample Preparation and Assay Protocols” by Boster antibody and ELISA experts. Kurien , B. T., & Scofield, R. H. (2006). Western blotting.  Methods ,  38 (4), 283-293. Fido, R. J., Tatham, A. S., & Shewry , P. R. (1995). Western blotting analysis.  Plant gene transfer and expression protocols , 423-437. Singh, N., Shepherd, K., & Cornish, G. (1991). A simplified SDS-PAGE procedure for separating.  J. Cereal Sci ,  14 , 203-208. Simpson, R. J. (2006). SDS-PAGE of proteins.  Cold Spring Harbor Protocols ,  2006 (1), pdb-prot4313. 45

46. Thank you 46