Transfer blotting

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

In order to facilitate analysis of FeBABE produced fragments, the prey protein or biomolecule is labeled at one end with a tag that can be detected after electrophoresis, usually in a transfer blot. The tag can be a fusion tag, such as 6X His, or any other group that can be targeted with an antibody and detected. Alternatively, radiolabels and fluorescent labels have been used with prey molecules, including the use of end-labeled DNA to study where DNA binding proteins dock onto the oligonucleotide sequence. 

Transfer (blotting) of DNA to membranes was introduced by E.M. Southern in 1974, and as a pun the transfer of RNA as the opposite of DNA was named Northern blot and that of proteins got the direction West. 

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. 

Immunoblotting techniques involve the identification of a protein target via antigen-antibody-specific reactions. Proteins are typically separated by electrophoresis in polyacrylamide gels, and then transferred ( blotted ) onto chemically resilient membranes (e.g., nitrocellulose, polyvinylidene difluoride) where they bind in the pattern they took in the gel. The membrane is overlaid with a primary antibody directed to the specific target, then with a secondary antibody (anti-immunoglobulin) labeled with radioisotopes, enzymes or other marker compounds. 

Protein samples are separated by one-dimensional SDS-PAGE or two-dimensional gel electrophoresis in polyacrylamide gels. The separated proteins are then transferred (blotted) to a nitrocellulose or nylon sheet. This is incubated with specific antibody to the protein and then unbound antibody is washed away. Those proteins in the gel that bind the antibody are detected either by autoradiography (if the specific antibody was radiolabeled) or by using a second labeled antibody that binds to the primary antibody. 

Methods used in EIH may be extended to other techniques where identification and localization of antigens are important the protein and nucleic acid transfer blots (Chapter 16) may be subjected to these methods. As with EIA, epitopes rather than antigens or molecules are recognized by EIH methods. Shared reactivity and crossreactivity (Section 8.6) may, therefore, interfere as in the AA-type assays (Section 2.3). 

Positive (+) and negative (-) symbols denote reactivity or lack of reactivity, respectively, of lectin reagent with the region of an SDS-PAGE/transfer blot corresponding to PGase. 

DNA is denatured in the gel (RNA is denatured before electrophoresis) and the face of the gel slab is placed in contact with a membrane made of nitrocellulose, nylon, or another material. The single-stranded nucleic acid molecules are then transferred from the gel to the membrane by capillary transfer (blotting) or other methods. The membrane containing the blotted nucleic acids is called the blot. 

In many cases, for the purpose of protein identification, immunochemical techniques have been combined with separation methods and with other methodologies. A good example of a combined procedure is the identification of the protease-resistant pathological form of the prion protein, in which the products of specific proteolysis of the whole tissue are first separated by electrophoresis, and identified by specific monoclonal antibodies after transfer (blotting) to a suitable membrane. Another common application of immunoblotting techniques is the identification of the food proteins (and of peptides... 

Southern Developed gel-transfer blotting technique for detection of specific DNA sequences... 

Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

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. 

Western blotting A means of transferring protein bands from an electrophoresis gel onto a fixing medium for further analysis. 

Fig. 2. Time course of accumulation of HSP mRNA. One jUg of poly(A) RNA isolated from soybean hypocotyls after different times of incubation at 42.5 °C (hs) or at additional times after transfer back to 28 °C after 4 h at the elevated temperature (recovery), were electrophoresed in formaldehyde agarose gels. Blots of these gels were hybridised with a mixture of four cDNAs encoding small soybean HSPs ranging from 15 to 23 kDa. From Schoffl Key (1982).

If the genetic lesion is understood and a specific probe is available, prenatal diagnosis is possible. DNA from cells collected from as little as 10 mL of amniotic fluid (or by chorionic villus biopsy) can be analyzed by Southern blot transfer. A fetus with the restriction pattern AA in Figure 40-10 does not have sickle cell disease, nor is it a carrier. A fetus with the SS pattern will develop the disease. Probes are now available for this type of analysis of many genetic diseases. 

Northern blot A method for transferring RNA from an agarose gel to a nitrocellulose filter, on which the RNA can be detected by a suitable probe. 

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. 

WN. Burnette (1981) "Western Blotting" Electrophoretic transfer of proteins from Sodium Dodecyl Polyacrylamide gels to unmodified nitrocellulose and radiographic... 

Specificity of the antisera was assessed by Western blotting. Electrophoretically separated proteins from culture filtrates were transferred to 0.45 fim nitrocellulose membranes. After transfer of proteins, membranes were... 

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