Immunoblotting techniques-ELISA, Western blotting, Southern blotting

Pharmaceutical Biotechnology, BP605T, Western blotting, Southern blotting, ELISA, Immunoblotting techniques, nitrocellulose membrane, horse-radish peroxidase, radioisotope, autoradiography, enzyme-substrate reaction, gel electrophoresis, Bovine serum albumin, preparation of gel for gel electrophoresis, TAE, TBE, SDS-PAGE electrophoresis, blotting techniques, polyacrylamide gel, PVDF, TBS-T, chemiluminescence, Antigen, antibody, spectrophotometer

1. WESTERN BLOTTING, ELISA, SOUTHERN BLOTTING Presented by:- STEFFI THOMAS Assistant Professor School of Pharmacy, LNCTU

2. Gel electrophoresis is a technique commonly used in laboratories to separate charged molecules like DNA, RNA and proteins according to their size. Charged molecules move through a gel when an electric current is passed across it. An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge. GEL ELECTROPHORESIS

3. The movement of charged molecules is called migration. Molecules migrate towards the opposite charge. A molecule with a negative charge will therefore be pulled towards the positive end (opposites attract!). The gel consists of a permeable matrix, a bit like a sieve, through which molecules can travel when an electric current is passed across it. Smaller molecules migrate through the gel more quickly and therefore travel further than larger fragments that migrate more slowly and therefore will travel a shorter distance. As a result the molecules are separated by size.

4. Electrophoresis enables you to distinguish DNA fragments of different lengths. DNA is negatively charged, therefore, when an electric current is applied to the gel, DNA will migrate towards the positively charged electrode. Shorter strands of DNA move more quickly through the gel than longer strands resulting in the fragments being arranged in order of size. The use of dyes, fluorescent tags or radioactive labels enables the DNA on the gel to be seen after they have been separated. They will appear as bands on the gel. A DNA marker with fragments of known lengths is usually run through the gel at the same time as the samples. By comparing the bands of the DNA samples with those from the DNA marker, you can work out the approximate length of the DNA fragments in the samples.

5. PREPARATION OF GEL :- Agarose gels are typically used to visualise fragments of DNA. The concentration of agarose used to make the gel depends on the size of the DNA fragments you are working with. Agarose gels are used for the electrophoresis of  DNA ,  RNA  and  Protein. It has got a Horizontal gel apparatus. The higher the agarose concentration, the denser the matrix and vice versa. Smaller fragments of DNA are separated on higher concentrations of agarose whilst larger molecules require a lower concentration of agarose . To make a gel, agarose powder is mixed with an electrophoresis buffer ( TAE-  ( Tris -acetate-EDTA) or TBE-  ( Tris -borate-EDTA))and heated to a high temperature until all of the agarose powder has melted. The molten gel is then poured into a gel casting tray and a “comb” is placed at one end to make wells for the sample to be pipetted into.

6. Once the gel has cooled and solidified (it will now be opaque rather than clear) the comb is removed. Many people now use pre-made gels. The gel is then placed into an electrophoresis tank and electrophoresis buffer is poured into the tank until the surface of the gel is covered. The buffer conducts the electric current. The type of buffer used depends on the approximate size of the DNA fragments in the sample. A dye is added to the sample of DNA prior to electrophoresis to increase the viscosity of the sample which will prevent it from floating out of the wells and so that the migration of the sample through the gel can be seen.

7. The prepared DNA samples are then pipetted into the remaining wells of the gel. When this is done the lid is placed on the electrophoresis tank making sure that the orientation of the gel and positive and negative electrodes is correct (we want the DNA to migrate across the gel to the positive end). The electrical current is then turned on so that the negatively charged DNA moves through the gel towards the positive side of the gel. Shorter lengths of DNA move faster than longer lengths so move further in the time the current is run. The distance the DNA has migrated in the gel can be judged visually by monitoring the migration of the loading buffer dye.

8. SDS-PAGE Electrophoresis:- -Sodium Dodecyl Sulfate (SDS) - polyacrylamide gel  electrophoresis is routinely used for the separation of proteins on the basis of their mass. -It involves the use of vertical gel apparatus to separate proteins. - Buffer system  the separation and migration patterns of proteins in gel electrophoresis are determined by the chemical composition and pH of the buffer system.

9. Blot means a dark mark/stain It is a procedure in which a protein or nucleic acid is separated on a gel are then transferred directly to an immobilizing medium for identification. Blots are techniques for transferring DNA , RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis. What is blotting?

10. Types of blotting techniques

11. A technique for detecting specific proteins in a given sample of tissue homogenate or extract which is separated by electrophoresis by use of labeled antibodies. So called since it has some similarity to a Southern blot. WESTERN BLOT ( Immunoblotting )

12. Protein blotting is an analytical method that involves the immobilization of proteins on membranes before detection using monoclonal or polyclonal antibodies Protein blotting

13. Western blotting is an Immunoblotting technique which rely on the specificity of binding between a molecule of interest and a probe to allow detection of the molecule of interest in a mixture of many other similar molecules. In Western blotting, the molecule of interest is a protein and the probe is typically an antibody raised against that particular protein. The SDS PAGE (Sodium Dodecyl / lauryl Sulfate - Polyacrylamide Gel Electrophoresis) technique is a prerequisite for Western blotting . Principle of Western Blotting

14. SDS (has long aliphatic chain + sulfate group)- is a protein separator i.e. it is an anionic detergent. (1)It denatures proteins. (2)It is used in denaturing polyacrylamide gel electrophoresis for the determination of protein molecular weight. (3)This detergent interacts with denatured protein to form strong negatively charged complex.

15. Western blot analysis can analyze any protein sample whether from cells or tissues, but also can analyze recombinant proteins synthesized in vitro . Western blot is dependent on the quality of antibody you use to probe for your protein of interest, and how specific it is for this protein This method is used in the fields of molecular biology, biochemistry, immunogenetics and other molecular biology disciplines. Advantages of Western Blot

16. Western blotting is a more specific test for detection of HIV. It can detect one protein in a mixture of proteins while giving information about the size of the protein and so is more specific. Western blot test is referred to as the Gold Standard It also tells you how much protein has accumulated in cells.

17. If a protein is degraded quickly, Western blotting won’t detect it well. This test takes longer than other existing tests. It might also be more costly DISADVANTAGES

18. Tissue preparation Gel electrophoresis Transfer Blocking Detection Analysis Procedure

19. We will introduce an antibody to the target protein (particular protein) in electrophoresis so that the interaction occurs. If we use 1 antibody it is called primary antibody that binds to target protein. If we use 2 antibodies, it is called secondary antibody that binds to the primary antibody .

20. Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender. It should be noted that bacteria, virus or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Tissue preparation is often done at cold temperatures to avoid protein denaturing. 1.TISSUE PREPARATION

21. Resected tissue in pre-cooled (4ºC) normal saline to wash off any blood from the tissue Chop the tissue into small pieces (0.1-1g each) after weighing Add protease enzyme Mince the tissue and place the minced tissue in the tissue homogenizer. Grind the tissue until fully homogenized. Add ice-cold lysis buffer (e.g. for 5 mg piece of tissue, add 300 μ l of buffer. Buffer volume should be determined in relation to the amount of tissue present)

22. After ultrasonication , this forms “ Tissue homogenate ” Lyse it on ice for 4-5 hours or at 4ºC (high speed) for 5 min Sonication Centrifugation at 10000 rpm at 4ºC for 10 min Discard lipid (at top) and cell debris (at bottom). Take out protein solution in middle and put it in a fresh tube

23. Mix the protein solution with SDS-PAGE loading buffer Denature the solution in 100ºC water bath for 5 min. The solution can be used immediately or stored at -20ºC for several months or at 4ºC for 1-2 weeks.

24. The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point molecular weight, electric charge, or a combination of these factors. The principle involved is the difference in the ELECTROPHORETIC MOBILITIES of different proteins. 2.GEL ELECTROPHORESIS

25. Uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed(detected) using antibodies specific to the target protein.

26. In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of supported nitrocellulose or Polyvinylidene Difluoride (PVDF). So a membrane made up of supported nitrocellulose (it can withstand autoclave temperature and retains the ease of wetting and protein binding features of nitrocellulose) or PVDF is used. PVDF membrane must be wetted in 100% methanol prior to use, otherwise protein will not stick onto it due to the presence of SDS. 3.TRANSFER

27. The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or supported nitrocellulose membrane.

28. The membrane has the ability to bind to proteins in this case both the target and antibodies are proteins and so there could be some unwanted binding. A blocking buffer is a solution of irrelevant protein, mixture of proteins, or other compound that passively adsorbs to all remaining binding surfaces of the plate. It helps in reducing interference by other proteins other than target protein. Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein - typically TBS-T ( Tris -buffered saline with a minute percentage of detergent such as Tween 20) with Bovine Serum Albumin (BSA). This buffer is used for washing nitrocellulose membrane in western blotting 4.BLOCKING

29. The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. Thus, when the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein After this the membrane is incubated with 1º antibody specific to target protein.

30. During the detection process, the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme, which when exposed to an appropriate substrate drives a colorimetric reaction and produces a color. Then the membrane is incubated with HRP- labelled ( Horse Radish Peroxidase is the enzyme that enhance chemiluminescence ) 2º antibody specific to 1º antibody. The point where signal is produced by 2º antibody, is where the target protein is located. 5.DETECTION

31. After the unbound probes are washed away, the western blot is ready for detection of the probes that are labeled and bound to the protein of interest. Size approximations are taken by comparing the stained bands to that of the marker loaded during electrophoresis. Image is produced (black dots and bands are produced) Dark bands- high concentration Faint band- lowest concentration 6.ANALYSIS

32. To detect HIV To detect BSE (Bovine Spongiform Encephalopathy) (Mad cow disease) Confirmatory test for Hepatitis B infection Applications

33. A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples. The Southern Blot allows the visualization of one DNA fragment from a whole genome DNA extract. Southern blotting combines transfer of electrophoresis -separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named after its inventor, the British biologist Edwin Mellor Southern . SOUTHERN BLOTTING

34. The key to this method is hybridization. Hybridization : It is the process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target DNA. There are 2 important features of hybridization: • The reactions are specific-the probes will only bind to targets with a complementary sequence. • The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules. Principle of southern blotting

35. DNA-DNA hybridization :- Uses gel electrophoresis together with hybridization probes to characterize restriction fragments of genomic DNA (or DNA from other sources, such as plasmids)

36. Steps

37. Step 1:-Extract and purify DNA from cells •Isolate the DNA from the rest of the cellular material in the nucleus. •Incubate specimen with detergent to promote cell lysis . • Lysis frees cellular proteins and DNA. •Proteins are enzymatically degraded by incubation with proteinase . •Organic or non-inorganic extraction removes proteins. •DNA is purified from solution by alcohol precipitation. •Visible DNA fibers are removed and suspended in buffer. DNA is restricted with enzymes (restriction endonuclease ).

38. Step 2:-Gel Electrophoresis Separates DNA fragments according to size Denature DNA- - The restriction fragments present in the gel are denatured with alkali. -This causes the double stranded to become single-stranded. - DNA is then neutralized with NaCl to prevent re-hybridization before adding the probe.

39. Step 4:-Hybridization •Incubate nitrocellulose sheet with a minimal quantity of solution containing 32 P-labeled ssDNA probe. •Probe sequence is complementary to the DNA of interest. •Incubate for several hours so that probe can anneal to its target sequence(s). •Wash & dry nitrocellulose sheet.

40. Step 5:-Autoradiography •Place nitrocellulose sheet over X- ray film. •X-ray film darkens where the fragments are complementary to the radioactive probes. If the probe is radioactive, the particles emits when expose to X-ray film.  

41. Diagnosis and detection of genetic diseases such as sickle cell anaemia Used in forensics (DNA fingerprinting) To identify specific DNA in a DNA sample Identify mutations, deletions, and gene rearrangements. Prognosis of cancer and in prenatal diagnosis of genetic diseases. Applications

42. It is a very sensitive technique to detect the presence of antigen or antibody in blood . It is similar to Radioimmuno Assay (RIA), but it uses radioisotopes to see the presence of antigen or antibody. In case of ELISA we use enzyme-substrate reaction and the colouration produced due to it (i.e. enzyme-substrate reaction) as a process of identifying antigen or antibody. ELISA (Enzyme Linked Immunosorbent Assay)

43. Antigen/antibody is adsorbed on to the plastic surface (‘ sorbent ’) Antigen is recognized by specific antibody (‘ immuno ’) This antibody is recognized by secondary antibody which has an enzyme attached (‘ enzyme-linked ’) Substrate reacts with the enzyme to produce a product, usually coloured . Why is it called “Enzyme-linked Immunosorbent Assay”?

44. As it is a very sensitive technique, it can detect protein even if it in picogram level. For e.g. if you have an infection (antigen) in your body due to any bacteria, fungi or virus, your body produces antibody which is specific against that particular infection. If we determine that particular antibody in your body, this means that there is infection in your blood serum.

45. Basic terms radish

46. Enzyme : Horse Radish Peroxidase (HRP), MW 44, 000, glycoprotein with 4 lysine residues. Substrate : TMB (3,3',5,5', tetramethylbenzidine ). The enzyme acts as a catalyst to oxidize substrate in the presence of Hydrogen peroxide to produce a blue color. Reaction stopped with dilute acid to cause complex to turn yellow.

47. 96 wells Microtiter plate (8×12) Requirements

48. Multipipette (8 channel 100 μ l pipette)

49. radish

50. 1. Antibody is coated inside the well 2. Antigen containing solution is added which binds to the antibody, after this washing is done to remove unbound antigens to be washed away 3.An antibody (2º antibody) specific to that antigen is added which is linked with an enzyme (depicted in circular red dots) 4. Substrate (Yellow coloured circle) is added, which reacts with enzyme to form a coloured poduct

51. General procedure

52. INDIRECT ELISA COMPETITIVE ELISA SANDWICH ELISA Types of ELISA

53. Indirect ELISA

54. Antibody can be detected or quantitatively determined with Indirect ELISA Serum or some other sample containing primary antibody (Ab 1 ) to an antigen-containing microtiter well and allowed to react with the antigen attached to the well. After any free Ab 1 is washed away, the presence of antibody bound to the antigen is detected by adding an enzyme-conjugated secondary antibody (Ab 2 ), which binds to primary antibody. Any free Ab 2 then is washed away and a substrate for the enzyme is added. The amount of coloured reaction product formed is measured by specialized spectrophotometric methods. It is so called because the enzyme is linked to secondary antibody and not the primary antibody.

55. Competitive ELISA

56. Another variation for measuring amount of antigen is competitive ELISA. In this technique, antibody is first incubated in solution with a sample containing antigen. The antigen-antibody mixture is then added to an antigen coated microtiter well. The more antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well. Addition of an enzyme-conjugated secondary antibdy (Ab 2 ) specific for the primary antibody can be used to determine the amount of primary antibody bound to the well as in indirect ELISA.

57. SANDWICH ELISA

58. Antigen can be detected or measured by sandwich ELISA. Here antibody (rather than antigen) is immobilized on microtiter well. A sample containing antigen is added and allowed to react with the immobilized antibody. After this the well is washed A second enzyme-linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen. After any free second antibody is removed by washing, substrate is added, and the colored reaction product is measured.

59. https://microbeonline.com/western-blot-technique-principle-procedures-advantages-and-disadvantages/ Punt Jenni , Stranford Sharon, “ Kuby Immunology”, Published by W.H. Freeman & Co Ltd., 8 th edition, Pg no. 148-154 https:// www.slideshare.net/sabaahmed56/elisa-ppt-5367857 REFERENCES