Blot transfer techniques

Probe A molecule used to detect the presence of a specific fragment of DNA or RNA in, for instance, a bacterial colony that is formed from a genetic library or during analysis by blot transfer techniques common probes are cDNA molecules, synthetic oligodeoxynucleotides of defined sequence, or antibodies to specific proteins. 

Northern blot The technique by which molecules of ribonucleic acid (RNA) are separated by gel electrophoresis, transferred to a membrane support, and incubated with labeled oligonucleotide probes. Specificity is obtained by using ohgonucleotide probes that have sequences complementary to the target RNA. 

Southern s technique could not be applied to the blot transfer of RNA separated by gel electrophoresis. Alwine et al. (A2) therefore devised a procedure in which RNA bands are blot transferred from the gel onto the chemically reactive paper, where they are bound covalently. The reactive paper is prepared by diazotization of aminobenzyl oxymethyl paper, which can be prepared from Whatman paper by a series of uncomplicated reactions. Once covalently bound, the RNA is available for hybridization with radiolabelled DNA probes. Hybridizing bands are detected autoradiographically. This method is termed Northern blotting. 

Soudiem blotting a technique used for transferring DNA from an agarose gel aJfter electrophoresis onto a membrane, such as one made of nitrocellulose (13.7) spacer region a region of eukaryotic DNA that is between nucleosomes (9.3)... 

Among the different gene transfer techniques, microinjection is by far the most efficient procedure. Only microinjection allows the transfer of a known number of test molecules either into the cytoplasm or into the nuclei of the recipient cell Up to 100% of the recipient cells support expression of the transferred material, and stable transformed cell lines can be isolated with a frequency of 20-30%. Biochemical studies can be performed with 50-100 injected cells, and the injected material (e.g., DNA, RNA) can be reisolated and further analyzed by standard techniques (e.g., Southern and Northern blots, electron microscopy) (for review, see Graessmann and Graessmann, 1983 Graessmann et al., 1983 Ceiis et al., 1986). 

Eor example, the technique of Southern blotting was developed (68) for use with agarose gel electrophoresis of DNA fragments. Southern blots are designed to detect specific sequences of DNA. After electrophoresis is complete, the DNA is denatured and the single stranded DNA transferred to the specially prepared nitrocellulose paper. The nitrocellulose is then incubated with radioactive RNA or DNA complementary to those DNA sequences of interest. After the nitrocellulose has been sufftciendy incubated with the radioactive complementary DNA, autoradiography is used to identify the fragments of interest. 

Transfer (blot) gel to nitrocellulose filter using Soudiern blot technique... 

After the initial separation by a conventional electrophoretic technique in a gel, the proteins are transferred (or blotted) electrophoretically from the gel to a membrane, usually nitrocellulose. The gel and the membrane, which has been previously soaked in a suitable electrophoretic buffer, are sandwiched between two electrodes. A voltage is applied, e.g. 100 V, and the proteins migrate from... 

Another similar technique to Southern blotting is Northern blotting. Here, instead of DNA fragments, mRNA fragments are probed with a labelled cDNA probe after separation by electrophoresis and transfer to nitrocellulose membranes. Northern blotting is used to detect and quantify mRNA from tissue extracts. 

Proteins bound to the surfaces of synthetic membranes retain their antigenicity and are accessible to antibody probes. The most common membrane-based immunoassay technique is called immunoblotting or, more popularly, Western blotting. In Western blotting, proteins are transferred from an electrophoresis gel to a... 

Transfer by blotting to a nylon membrane allows the heat treatment involved in hybridisation to take place. Southern blotting technique. 

Southern blotting is a technique that can be used to detect specific genes present in DNA. The DNA is cleaved using a restriction endonuclease, the pieces are separated by gel electrophoresis and then transferred to a nitrocellulose membrane for analysis. The fragment of interest is detected using a probe. 

One way to select a desired segment of DNA from a digest of chromosomal DNA is to sort out the "restriction fragments" by gel electrophoresis.613 The DNA from the gel can be transferred to a nitrocellulose sheet while retaining the separation pattern using a method devised by Southern.560 614 615 In this Southern blot technique, solvent flows from a pool beneath the gel up through the gel and the nitrocellulose sheet into paper towels. The DNA is trapped on the nitrocellulose in the same pattern observed in the electrophero-gram. A suitably labeled probe such as cDNA with... 

Western blotting has become an important, modern technique for analysis and characterization of proteins. The procedure consists of, first, the electrophoretic transfer (blotting) of proteins from polyacrylamide gels to synthetic membranes. The transferred blots are then probed using immunological detection methods to identify proteins of specific structure and/or function. In this experiment, bovine serum will be fractionated by SDS-PAGE and the proteins blotted onto a nitrocellulose membrane. Serum glycoproteins will be identified by their specific interaction with the lectin concanavalin A. 

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