1. Midterm Exam Wednesday, October 20. 10 questions 8 questions will be taken from the class presentations, 2 questions will be for a general understanding Evaluation criteria Each question brings 4 points Maximal score for the exam – 40 points

2. An example of a question Q: lists methods of denaturating precipitation of proteins A: Precipitation with acetone or trifluoroacetic acid

3. Gel electrophoresis… going farther… Western blotting

4. Immunodetection A technique aimed at detection, quantification, and localization of antigens by means of antibody binding Quantitation of an antigen - Enzyme-linked immunosorbent assays Identification and characterization of protein antigens - Immunoprecipitation - Western blotting

5. Western blotting blot is an english word meaning this (In Western blotting, proteins separated by gel-electrophoresis are visualized after developing in a form of blots.)

6. Western blotting Western blotting is a tool to identify and quantify a specific protein in a complex mixture. The technique enables indirect detection of protein samples immobilized on a nitrocellulose or polyvinylidene fluoride (PVDF) membrane.

7. Western blotting Western blot starts from the separation of proteins by means of SDS- PAGE

8. Western blotting Then proteins are transferred to a membrane and an uncovered surface of the membrane is blocked with help of a non-relevant protein.

9. Electrophoretic transport unit

10. Western blotting The membrane is probed with a primary antibody. This antibody was raised against the target antigen.

11. Antigen is a molecule recognized by the immune system. The term came from antibody generator and was a molecule that binds specifically to an antibody. Antibodies are proteins that are produced by the immune system as a response to a foreign object, such as virus or bacteria Interaction of an antigen and an antibody is of a “lock and key” type. Each antibody binds to a specific antigen

12. Western blotting The membrane is washed and incubated with an enzyme-conjugated secondary antibody that is reactive towards the primary antibody. secondary antibody enzyme

13. Western blotting The membrane is washed again and is incubated with an appropriate enzyme substrate. The signal is either visually evaluated, if a colorimetric substrate was used, or is detected with X-ray film or imaging instrumentation for chemiluminescence and fluorescence

14. Steps of western blocking 1.Transfer of proteins from a gel plate to a membrane 2.Blocking 3.Primary antibody 4.Enzyme- conjugated secondary antibody 5.Attachment of a substrate 6.Reading of the signal

15. Steps of western blocking 1.Transfer of proteins from a gel plate to a membrane 2.Blocking 3.Primary antibody 4.Enzyme- conjugated secondary antibody 5.Attachment of a substrate 6.Reading of the signal

16. Blocking The blocking step is necessary to prevent the interaction of a primary antibody with a free surface of the membrane BSA non-fat dry milk usual non-relevant proteins used for blocking

17. Steps of western blocking 1.Transfer of proteins from a gel plate to a membrane 2.Blocking 3.Primary antibody 4.Enzyme- conjugated secondary antibody 5.Attachment of a substrate 6.Reading of the signal

18. Steps of western blocking 1.Transfer of proteins from a gel plate to a membrane 2.Blocking 3.Primary antibody 4.Enzyme- conjugated secondary antibody 5.Attachment of a substrate 6.Reading of the signal

19. Enzyme-conjugated secondary antibody Secondary antibody must be specific to the whole class of immunoglobulins Enzyme conjugate Alkaline phosphatase (AP, 140 kDa) Horseradish peroxidaze (HRP, 40 kDa) Common enzyme conjugates

20. Steps of western blocking 1.Transfer of proteins from a gel plate to a membrane 2.Blocking 3.Primary antibody 4.Enzyme- conjugated secondary antibody 5.Attachment of a substrate 6.Reading of the signal

21. Substrate Substrates for colorimetric detection AP 5-bromo-4-chloro-3’-indolylphosphate-p-toluidine salt (BCIP) nitro-blue tetrazolium chloride (NBT) naphthol AS-MX phosphate + Fast Red TR Salt (Fast Red) HRP 3,3’,5,5’-tetramethylbenzidine (TAB) 4-chloro-1-naphthol (4-CN) 3,3’-diaminobenzidine tetrahydrochloride (DAB)

22. Colorimetric detection Detection of mouse IgG Conjugated enzyme: AP AP Substrate: FirePhos BCIP/NBT

23. Substrate Substrates for chemiluminiscent detection Luminol (c) A. Alegria-Schaffer et al., Guide to protein purification, 2 nd Ed. Ch. 33

24. Chemiluminiscent detection Visualization of the signal charged-coupled device (CCD)

25. Substrate Substrates for fluorescent detection Western blotting detection via fluorescence is typically performed when there are two different targets on a single blot and high sensitivity is required. fluorescein rhodamine amino-methyl-coumarin-acetate Traditional fluorophores

26. Fluorescent detection Visualization of signal

27. Automation of Western blotting

28. Southern and Northern blotting Methods of the detection of specific DNA (Southern blotting) and RNA (Northern blotting) The Southern blot method was named after Edwin Southern, who invented the method in 1974

29. Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments.endonucleases The DNA fragments are then electrophoresed on an agarose gel to separate them by size.electrophoresedagarose gel If some of the DNA fragments are larger than 15 kb, then prior to blotting, the gel may be treated with an acid, such as dilute HCl, which depurinates the DNA fragments, breaking the DNA into smaller pieces, thus allowing more efficient transfer from the gel to membrane.kbHCldepurinates If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged DNA to a positively charged membrane, separating it into single DNA strands for later hybridization to the probe (see below), and destroys any residual RNA that may still be present in the DNA. The choice of alkaline over neutral transfer methods, however, is often empirical and may result in equivalent results. [citation needed]sodium hydroxidehybridizationcitation needed A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of (or below, depending on the direction of the transfer) the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. If transferring by suction 20X SSC buffer is used to ensure a seal and prevent drying of the gel. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel on to the membrane; ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane.nitrocellulosenylonmembrane20X SSCcapillary actionwater potentialion exchange The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours (standard conditions; nitrocellulose or nylon membrane) or exposed to ultraviolet radiation (nylon membrane) to permanently attach the transferred DNA to the membrane.ultraviolet radiation The membrane is then exposed to a hybridization probe—a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined. The probe DNA is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA. To ensure the specificity of the binding of the probe to the sample DNA, most common hybridization methods use salmon or herring sperm DNA for blocking of the membrane surface and target DNA, deionized formamide, and detergents such as SDS to reduce non- specific binding of the probe.hybridization proberadioactivityfluorescentformamideSDS After hybridization, excess probe is washed from the membrane (typically using SSC buffer), and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used.SSC bufferX-rayautoradiography Nucleic acid blotting by example of RNA (Northern blot) 1. Process starts from the separation of nucleic acid fragments by gel-electrophoresis. Agarose gel is used.

30. Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments.endonucleases The DNA fragments are then electrophoresed on an agarose gel to separate them by size.electrophoresedagarose gel If some of the DNA fragments are larger than 15 kb, then prior to blotting, the gel may be treated with an acid, such as dilute HCl, which depurinates the DNA fragments, breaking the DNA into smaller pieces, thus allowing more efficient transfer from the gel to membrane.kbHCldepurinates If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged DNA to a positively charged membrane, separating it into single DNA strands for later hybridization to the probe (see below), and destroys any residual RNA that may still be present in the DNA. The choice of alkaline over neutral transfer methods, however, is often empirical and may result in equivalent results. [citation needed]sodium hydroxidehybridizationcitation needed A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of (or below, depending on the direction of the transfer) the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. If transferring by suction 20X SSC buffer is used to ensure a seal and prevent drying of the gel. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel on to the membrane; ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane.nitrocellulosenylonmembrane20X SSCcapillary actionwater potentialion exchange The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours (standard conditions; nitrocellulose or nylon membrane) or exposed to ultraviolet radiation (nylon membrane) to permanently attach the transferred DNA to the membrane.ultraviolet radiation The membrane is then exposed to a hybridization probe—a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined. The probe DNA is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA. To ensure the specificity of the binding of the probe to the sample DNA, most common hybridization methods use salmon or herring sperm DNA for blocking of the membrane surface and target DNA, deionized formamide, and detergents such as SDS to reduce non- specific binding of the probe.hybridization proberadioactivityfluorescentformamideSDS After hybridization, excess probe is washed from the membrane (typically using SSC buffer), and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used.SSC bufferX-rayautoradiography Nucleic acid blotting by example of RNA (Northern blot) 2. Transfer of separated nucleic acid fragments to membrane Nitrocellulose or nylon membrane are used. Transfer is performed by capillary forces

31. Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments.endonucleases The DNA fragments are then electrophoresed on an agarose gel to separate them by size.electrophoresedagarose gel If some of the DNA fragments are larger than 15 kb, then prior to blotting, the gel may be treated with an acid, such as dilute HCl, which depurinates the DNA fragments, breaking the DNA into smaller pieces, thus allowing more efficient transfer from the gel to membrane.kbHCldepurinates If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged DNA to a positively charged membrane, separating it into single DNA strands for later hybridization to the probe (see below), and destroys any residual RNA that may still be present in the DNA. The choice of alkaline over neutral transfer methods, however, is often empirical and may result in equivalent results. [citation needed]sodium hydroxidehybridizationcitation needed A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of (or below, depending on the direction of the transfer) the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. If transferring by suction 20X SSC buffer is used to ensure a seal and prevent drying of the gel. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel on to the membrane; ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane.nitrocellulosenylonmembrane20X SSCcapillary actionwater potentialion exchange The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours (standard conditions; nitrocellulose or nylon membrane) or exposed to ultraviolet radiation (nylon membrane) to permanently attach the transferred DNA to the membrane.ultraviolet radiation The membrane is then exposed to a hybridization probe—a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined. The probe DNA is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA. To ensure the specificity of the binding of the probe to the sample DNA, most common hybridization methods use salmon or herring sperm DNA for blocking of the membrane surface and target DNA, deionized formamide, and detergents such as SDS to reduce non- specific binding of the probe.hybridization proberadioactivityfluorescentformamideSDS After hybridization, excess probe is washed from the membrane (typically using SSC buffer), and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used.SSC bufferX-rayautoradiography Nucleic acid blotting by example of RNA (Northern blot) 3. Fixation of nucleic acids on the membrane by heating or UV irradiation

32. Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments.endonucleases The DNA fragments are then electrophoresed on an agarose gel to separate them by size.electrophoresedagarose gel If some of the DNA fragments are larger than 15 kb, then prior to blotting, the gel may be treated with an acid, such as dilute HCl, which depurinates the DNA fragments, breaking the DNA into smaller pieces, thus allowing more efficient transfer from the gel to membrane.kbHCldepurinates If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged DNA to a positively charged membrane, separating it into single DNA strands for later hybridization to the probe (see below), and destroys any residual RNA that may still be present in the DNA. The choice of alkaline over neutral transfer methods, however, is often empirical and may result in equivalent results. [citation needed]sodium hydroxidehybridizationcitation needed A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of (or below, depending on the direction of the transfer) the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. If transferring by suction 20X SSC buffer is used to ensure a seal and prevent drying of the gel. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel on to the membrane; ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane.nitrocellulosenylonmembrane20X SSCcapillary actionwater potentialion exchange The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours (standard conditions; nitrocellulose or nylon membrane) or exposed to ultraviolet radiation (nylon membrane) to permanently attach the transferred DNA to the membrane.ultraviolet radiation The membrane is then exposed to a hybridization probe—a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined. The probe DNA is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA. To ensure the specificity of the binding of the probe to the sample DNA, most common hybridization methods use salmon or herring sperm DNA for blocking of the membrane surface and target DNA, deionized formamide, and detergents such as SDS to reduce non- specific binding of the probe.hybridization proberadioactivityfluorescentformamideSDS After hybridization, excess probe is washed from the membrane (typically using SSC buffer), and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used.SSC bufferX-rayautoradiography Nucleic acid blotting by example of RNA (Northern blot) 4. Labeling of analyzed RNA with a probe

33. Attachment of a probe to RNA membrane RNA of interest labeled probe complimentary oligonucleotide label

34. Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments.endonucleases The DNA fragments are then electrophoresed on an agarose gel to separate them by size.electrophoresedagarose gel If some of the DNA fragments are larger than 15 kb, then prior to blotting, the gel may be treated with an acid, such as dilute HCl, which depurinates the DNA fragments, breaking the DNA into smaller pieces, thus allowing more efficient transfer from the gel to membrane.kbHCldepurinates If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged DNA to a positively charged membrane, separating it into single DNA strands for later hybridization to the probe (see below), and destroys any residual RNA that may still be present in the DNA. The choice of alkaline over neutral transfer methods, however, is often empirical and may result in equivalent results. [citation needed]sodium hydroxidehybridizationcitation needed A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of (or below, depending on the direction of the transfer) the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. If transferring by suction 20X SSC buffer is used to ensure a seal and prevent drying of the gel. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel on to the membrane; ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane.nitrocellulosenylonmembrane20X SSCcapillary actionwater potentialion exchange The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours (standard conditions; nitrocellulose or nylon membrane) or exposed to ultraviolet radiation (nylon membrane) to permanently attach the transferred DNA to the membrane.ultraviolet radiation The membrane is then exposed to a hybridization probe—a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined. The probe DNA is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In some cases, the hybridization probe may be made from RNA, rather than DNA. To ensure the specificity of the binding of the probe to the sample DNA, most common hybridization methods use salmon or herring sperm DNA for blocking of the membrane surface and target DNA, deionized formamide, and detergents such as SDS to reduce non- specific binding of the probe.hybridization proberadioactivityfluorescentformamideSDS After hybridization, excess probe is washed from the membrane (typically using SSC buffer), and the pattern of hybridization is visualized on X-ray film by autoradiography in the case of a radioactive or fluorescent probe, or by development of color on the membrane if a chromogenic detection method is used.SSC bufferX-rayautoradiography Nucleic acid blotting by example of RNA (Northern blot) 5. Visualisation