western blotting 2Word文档格式.docx

1、Background and Introduction BackgroundAfter electrophoresis, only a minute amount of protein or nucleic acid is present in bands on electropherograms. In spite of this, there is often a need to extract the desired biomolecule from the gel for further investigation. This sometimes involves the tediou

2、s and cumbersome process of crushing slices of the gel in a buffer to release the trapped proteins or nucleic acids. Techniques are now available for removing nucleic acids and proteins from gels and characterizing them using probes to detect certain structural features or functions. After electroph

3、oresis, the biomolecules are transferred or “blotted” out of the geol onto a nitrocellulose filter or nylon membrane. The desired biomolecule is now accessible on the filter for further analysis. The first blotting technique was reported by E.Southern in 1975. Using labelled complementary DNA probes

4、, he searched for certain nucleotide sequences among DNA molecules blotted from the gel. This technique of detecting DNA-DNA hybridization is called Southern blotting. The general blotting technique has now been extended to the transfer and detection of specific RNA with labelled complementary DNA p

5、robes （Northern blotting） and the transfer and detection of proteins that react with specific antibodies （Western blotting）. In practice, the electropherogram is alkali treated, neutralized, and placed in contact with the filter or nylon membrane. A buffer is used to facilitate the transfer. The loc

6、ation of the desired nucleic acid or protein is then detected by incubation of the membrane with a radio labelled probe and autoradiography, by use of a biotinylated probe or by linkage to an enzyme-catalyzed reaction that generates a colour.Blotting techniques have many applications, including mapp

7、ing the genes responsible for inherited diseases by using restriction fragment length polymorphisms （PFLPs）, screening collections of cloned DNA fragments （DNA libraries）, “DNA fingerprinting” for analysis of biological material remaining at the identification of specific proteins.In this experiment

8、, western blotting is employed to identify the specific protein on the electrophoresis gel. IntroductionIn experiment four, we have tried the technique of SDS-PAGE. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis （SDS-PAGE） is the electrophoretic techniques applied to the measurement of th

9、e molecular weights of biological molecules. If the protein samples are treated so that they have a uniform charge, electrophoretic mobility then depends primarily on size. The molecular weights of proteins may be estimated if they are subjected to electrophoresis in the presence of a detergent, sod

10、ium dodecyl sulphate （SDS）, and a disulfide bond reducing agent, mercaptoethanol. This method is often called “denaturing electrophoresis.”. SDS-PAGE is valuable for estimating the molecular weight of protein subunits. This modification of gel electrophoresis finds its greatest use in charactering t

11、he sizes and different types of subunits in oligomeric proteins. SDS-PAGE is limited to a molecular weight range of 10,000 to 200,000. Gels of less than 2.5% acrylamide must be used for determining molecular weights above 200,000, but these gels do not set well and are very fragile because of minima

12、l cross-linking. A modification using gels of agarose acrylamide mixtures allows the measurement of molecular weights above 200,000.In SDS-PAGE, after running and staining with the dye Coomassie Blue, deeply colored bands appeared on the gel wherever there was a protein. If molecular weight standard

13、s were included on the gel, it is feasible to estimate the molecular weight for a specific protein present on the gel. SDS-PAGE is indeed a very effective analytical tool to achieve fractionation of protein mixtures, to analyze purity, and to estimate molecular weight, but it provides no experimenta

14、l data to prove the identity of any of the dyed protein bands. A Coomassie Blue stain simply indicates the presence and location of each and every protein on the gel. It is often possible to identify proteins by treating gel bands directly with chemical reagents that react with a specific protein. F

15、or example, the identity and location of an enzyme may be noted by treating the gel with a substrate that is converted to a coloured product by enzyme. However, proteins are deeply embedded in the polyacrylamide gel matrix and are not readily accessible to most analytical reagents. This hinders spec

16、ific analysis of the protein bands in order to identify individual proteins. Proteins separated by PAGE may be transferred （or blotted） from the gel to a thin support matrix, usually a nitrocellulose membrane, which strongly binds and immobilizes proteins. The protein blots on the membrane surface a

17、re more accessible to chemical or biochemical reagents for further analysis. When the transfer process is coupled with protein identification using highly specific and sensitive immunological detection techniques, the procedure is Western Blotting. Western blotting or immunoblotting assays of protei

18、ns have many advantages including the need for only small reagent volumes, short processing times, relatively inexpensive equipment, and ease of performance.To begin the Western blot procedure, a protein mixture for analysis and further characterization is fractionated by PAGE. Since denaturing, SDS

19、-PAGE results in better resolution than PAGE performed under native conditions, SDS-PAGE is usually preferred; however, the detection method used at the conclusion of the blotting experiment must be able to recognize denatured protein subunits. The next step involves selection of the membrane matrix

20、 for transfer. Three types of support matrices are available for use: nitrocellulose, nylon, and polyvinyl-difluoride （PVDF）. Nitrocellulose membranes, currently the most widely used supports, have a satisfactory protein binding capacity （100g/cm2）, but they display weak binding of proteins of molec

21、ular weights smaller than 14,000 and they are subject to tearing. Binding of proteins to nitrocellulose membranes is noncovalent, most likely hydrophobic. Nylon membranes are stronger than nitrocellulose and some have a binding capacity up to 450g/cm2. However, since they are cationic, they only wea

22、kly bind basic proteins. During detection procedures, nylon membrane often display high background colours, so it is difficult to visualize proteins of interest. PVDF membranes bind proteins strongly （125g/cm2） and, because of their hydrophobic nature, give light background colour after analysis. Fo

23、r overall general use in protein transfer and immunoblotting, nitrocellulose membranes are the most common choice, as we did in this experiment.The actual blotting process can be accomplished by one of the two methods below: passive （or capillary） transfer and electroblotting. In passive transfer, t

24、he membrane is placed in direct contract with the polyacrylamide gel and organized in a sandwich-like arrangement consisting of （from bottom to top） filter paper soaked with transfer buffer, gel, membrane, and more filter paper. The sandwich is compressed by a heavy weight. Buffer passes by capillar

25、y action from the bottom filter paper through the gel, transferring the protein molecules to the membrane, where the macromolecules are immobilized. Passive transfer is very time consuming, sometimes requiring 1-2 days for complete protein transfer. Faster and more efficient transfer is afforded by

26、the use of an electroblotter. Here a sandwich of filter paper, gel, membrane, and more filter paper is prepared in a cassette, which is placed between platinum electrodes. An electric current is passed through the gel, causing the proteins to electrophorese out of the gel and onto the membrane.Thus,

27、 the Western blot procedure is concluded by probing the blotted protein bands and detecting a specific protein or group of proteins among the blots. In other words, visualization of specific protein blots must be possible. The most specific identification techniques are based on immunology （antigen-

28、antibody） interactions. A general procedure for immunoblotting is outlined here:（1）. Proteins are transferred from electrophoresis gel to nitrocellulose membrane. Blocker proteins bind to unoccupied sites on the membrane. （2）. The membrane is incubated with a primary antibody directed against the pr

29、otein of interest. （3）. A secondary antibody is directed against the primary antibody.（4）. The second antibody is conjugated with an enzyme to provide a detection mechanism. Substrate solution is added to the blot. The conjugated enzyme catalyzes the conversion of substrate to product to form a colo

30、red precipitate at the site of the protein-antibody complex.Before the protein detection process can begin, it is necessary to block protein binding sites on the membrane that are not occupied by blotted proteins. This is essential because antibodies used to detect blotted proteins are also proteins

31、 and will bind to the still remaining on blotted membrane and interfere with detection procedures. Protein binding sites still remaining on blotted membranes may be blocked by treatment with solutions of casein （major protein in milk）, gelation, or bovine serum albumin. In this experiment, bovine se

32、rum albumin is used as blocker.The blotted membrane, with all protein binding sites occupied, can mow be treated with analytical reagents for detection of specific proteins. Typically, the blotted membrane is incubated with an antibody specific for the protein of interest. This is the primary antibo

33、dy, which is a protein of the immunoglobulin G （IgG） class. The primary antibody binds to the desired protein, forming an antigen-antibody complex. The interaction between the protein and its antibody dose not usually result in a visible signal. The blot is then incubated with a secondary antibody, which is directed agains