Blot: a spot or stain, especially of ink on paper.

Transcription

1 Blotting technique

2 Blot: a spot or stain, especially of ink on paper. 2/27 In molecular biology and genetics, a blot is a method of transferring proteins, DNA or RNA, onto a carrier (for example, a nitrocellulose,pvdf or nylon membrane). In many instances, this is done after a gel electrophoresis, transferring the molecules from the gel onto the blotting membrane, and other times adding the samples directly onto the membrane. There are a variety of methods that have been used for transferring, including diffusion transfer, capillary transfer, vacuum blotting transfer and electroelution.

3 Common blot methods are : Dot blot Slot blot Southern blot for DNA Northern blot for RNA Western blot for proteins Reverse Northern blot for RNA Southwestern blot for Protein-DNA Far-Western blot for Protein-Protein Eastern blotting for posttranslational modification Far-Eastern blot for Lipids, Drugs and Hormones 3/27

4 Southern blot for DNA Northern blot for RNA Western blot for proteins Dot blot 4/27

5 Southern blot for DNA 'Southern' hybridization named after Sir Edwin Southern Developed in /27

6 Goals of Southern Hybridization Immobilize DNA onto a permanent substrate Identify DNA sequence (gene) of interest 6/27

7 Depurination Denaturation Neutralization Transfer Imaging 7/27 Washing Pre-Hybridization and Hybridization

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9 Northern blot The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University. Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot, named for biologist Edwin Southern. George Stark James Alwine 9/27 David Kemp

10 10/27 Procedure

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12 Applications Northern blotting allows one to observe a particular gene's expression pattern between tissues, organs, developmental stages, environmental stress levels, pathogen infection, and over the course of treatment. The technique has been used to show overexpression of oncogenes and downregulation of tumor-suppressor genes in cancerous cells when compared to 'normal' tissue, as well as the gene expression in the rejection of transplanted organs. 12/27 BlotBase is an online database publishing northern blots. BlotBase has over 700 published northern blots of human and mouse samples, in over 650 genes across more than 25 different tissue types.

13 Disadvantages and Advantages Microarrays are quite commonly used and are usually consistent with data obtained from northern blots; however, at times northern blotting is able to detect small changes in gene expression that microarrays cannot. The advantage that microarrays have over northern blots is that thousands of genes can be visualized at a time, while northern blotting is usually looking at one or a small number of genes. Compared to RT-PCR, northern blotting has a low sensitivity, but it also has a high specificity which is important to reduce false positive results. The advantages of using northern blotting include the detection of RNA size, the observation of alternate splice products, the use of probes with partial homology, the quality and quantity of RNA can be measured on the gel prior to blotting, and the membranes can be stored and reprobed for years after blotting. 13/27

14 Western blot The method originated from the laboratory of George Stark at Stanford. The name Western blot was given to the technique by W. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. 14/27 George Stark W. Neal Burnette

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16 Sample preparation Bromophenol Blue Source tissue Microcentrifuge tube SDS Tris DTT HCl ddh2o Sample buffer Heater Vortex Sample 16/27

17 Gel electrophoresis Gel cassette Negative electrode chamber Anode buffer Cathode buffer Sample Anode Cathode Tank Power source Separate protein bonds 17/27

18 18/27 Transfer

19 Blocking & Detection Primary antibody horseradish peroxidase Conjugated secondary antibodies Blocking buffer Wash buffer PBST PBST Membrane with transferred proteins Incubation Incubation Membrane (ready to be visualized) 19/27

20 Chemiluminescent detection Chemiluminescence reagent film scanner Membrane (ready to be visualized) results Image station 20/27

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23 Medical diagnostic applications The confirmatory HIV test employs a Western blot to detect anti-hiv antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane as above. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary antihuman antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody. A Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease'). Some forms of Lyme disease testing employ Western blotting. Western blot can also be used as a confirmatory test for Hepatitis B infection. 23/27 In veterinary medicine, Western blot is sometimes used to confirm FIV+ status in cats.

24 Dot blot It represents a simplification of the northern blot, Southern blot, or western blot methods. In a dot blot the biomolecules to be detected are not first separated by chromatography. The technique offers significant savings in time, as chromatography or gel electrophoresis, and the complex blotting procedures for the gel are not required. It offers no information on the size of the target biomolecule. If two molecules of different sizes are detected, they will still appear as a single dot. 24/27 Dot blots therefore can only confirm the presence or absence of a biomolecule or biomolecules which can be detected by the DNA probes or the antibody.

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26 Principles and Techniques of Biochemistry and Molecular Biology,Keith Wilson and John Walker, Kambridge university press, Burnette W N. A. Anal. Biochem : Introduction to Western Blotting, Claire Moore,abds erot e c, Morphosys Uk Ltd, Services/Applications/Protein-Expression-and Analysis/Western- Blotting.htm /27

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