Blotting techniques

These slides include all the 3 blotting techniques i,e Southern,northern and western.Their principle,procedure and application.

1. BLOTTING TECHNIQUES BY: FARHA BANU MSC MICROBILOGY

2. SOUTHERN BLOTTING The technique was developed by E.M.Southern in 1975. The southern blot is used to detect the presence of a particular DNA fragment in a sample. The DNA detected can be a single gene or it can be apart of a larger piece of DNA such as viral genome.

3. PRINCIPLE The key to this method is hybridization. Hybridization - Process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target DNA. There are two 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 noncomplementary molecules.

4. General Scheme for Southern Blot

5. PROCEDURE 1 :DNA purification Isolate the DNA in question from the rest of the cellular material in the nucleus. Incubate specimen with detergent to promote cell lysis,cell lysis frees cellular protein and DNA. Proteins are enzymatically degraded by incubation with proteinase . DNA is purified from solution by alcohol precipitation. Visible DNA fibers are removed and suspended in buffer.

6. Step 2:RESTRICTION DIGEESTION Cut the DNA into different sized framents using restriction endonucleases (RE).

7. Step 3:Gel electrophoresis Nucleic acid has a net negative charge and will move from the left to right.The larger molecules are held up while the smaller ones move faster.This results in a separation by size. Gels are Agarose or polyacrylamide With microscopic pores. Gel is soaked in a buffer which Controls the size of the pores.

8. Gels can be stained with ethidium bromide,this cause DNA to fluoresce under UV light which permits photography of the gel. This will help us to know the exact migration of DNA strands and the quality of the RE digestion of the test DNA.

9. Step 3. Nitrocellulose Blot Cover gel with nitrocellulose paper…then… Cover nitrocellulose paper with thick layer of paper towels. Compress apparatus with heavy weight. ssDNA binds to nitrocellulose at same position it had on the gel. Vacum dry nitrocellulose at 80  C to permanently fix DNA in place or cross link (via covalent bonds) the DNA to the membrane.

10. 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 at suitable renaturation temperature that will permit probe to anneal to its target sequence(s). Wash & dry nitrocellulose sheet.

11. Step 5. Autoradiography Place nitrocellulose sheet over X-ray film. X-ray film darkens where the fragments are complementary to the radioactive probes.

12. General Scheme for Southern Blot

13. APPLICATIONS Used to diagnose sickle cell-anemia. Diagnosis for HIV and infectoius disease. To identify specific DNA in a DNA sample. To isolate desired DNA for construction of rDNA . Identify mutations,deletions,and gene rearrangements.

14. NOTHERN BLOTTING

15. Northern Blotting Northern blotting is a technique for detection of specific RNA sequences. Northern blotting was developed by James Alwine and George Stark at Stanford University (1979) and was named such by analogy to Southern blotting

16. Steps involved in Northern Blotting RNA is isolated from several biological samples (e.g. various tissues, various developmental stages of same tissue etc.)

17. STEPS 2.Sample’s are loaded on gel  and the RNA samples are separated according to their size on an agarose gel . The resulting gel following after the electrophoresis run. 

18. STEPS The gel is then blotted on a nylon membrane or a nitrocellulose filter paper by creating the sandwich arrangement. N ylon membrane with a positive charge is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them.

19. STEPS Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat.

20. STEPS The membrane is placed in a dish containing hybridization buffer with a labeled probe. Thus, it will hybridize to the RNA on the blot that corresponds to the sequence of interest.  5. The membrane is washed to remove unbound probe.

21. STEPS The labeled probe is detected via autoradiography or via a chemiluminescence reaction (if a chemically labeled probe is used).  In both cases this results in the formation of a dark band on an X-ray film. Now the expression patterns of the sequence of interest in the different samples can be compared.

22. NORTHERN BLOTTING

23. APPLICATIONS Is useful for revealing the size of the mRNA encoded by a gene. Study RNA degradation Study RNA splicing Study RNA half-life Often used to confirm and check transgenic / knockout mice (animals)

24. WESTERN BLOTTING

25. WESTERN BLOTTING Protein blotting is an analytical method that involves the immobilization of proteins on membranes before detection using monoclonal or polyclonalantibodies . There are different blotting protocols(dot blot, 2D blot); one of the most powerful is western blotting.

26. Principle of Western Blotting 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 technique is a prerequisite for Western blotting

27. Definition The Western Blot is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

28. Gel electrophoresis Uses gel electrophoresis (SDS PAGE) 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.

29. The procedure includes Tissue preparation Gel electrophoresis Transfer Blocking Detection Analysis

30. Tissue Preparations 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.

31. Gel Electrophoresis 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.

32. Transferring In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). 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.

33. Another method for transferring the proteins is called electro blotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane

34. BLOCKING The membrane supports used in Western blotting have a high affinity for proteins. Therefore, after the transfer of the proteins from the gel, it is important to block the remaining surface of the membrane to prevent nonspecific binding of the detection antibodies during subsequent steps. A variety of blocking buffers ranging from milk or normal serum t o highly purified proteins have been used to block free sites on a membrane. The blocking buffer should improve the sensitivity of the assay by reducing background interference and improving the signal to noise ratio.

35. Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein - typically Bovine serum albumin(BSA) with a minute percentage of detergent such as Tween 20.

36. DETECTION During the detection process, the membrane is "probed" for the protein of interest with a antibody that is primary antibody and incubated. Secondary antibody which is linked to a reporter enzyme is added, which when exposed to an appropriate substrate drives a colorimetric reaction and produces a color.

37. ANALYSIS 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. The membrane is then detected using the label antibody,usually with an enzyme such as horse radish peroxide(HRP),which is detected by the signal it produces corresponding to the position of the target protein.This signal is captured on a film which is usually developed in a dark room.

38. ADVANTAGES While ELISA being a non specific test, 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 is also used as the definitive test for Bovine spongiform encephalopathy (BSE( Some forms of Lyme disease testing employ Western blotting. It also tells you how much protein has accumulated in cells.

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

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