Southern blot transfer procedure

Fig. 1. Southern blotting. The procedure shown is the original method of Southern using capillary action to blot the DNA bands from the gel to the nitrocellulose membrane. Electrolytic transfer is now often used instead.

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. 

The southern transfer procedure has eilso been extended to RNA now. The nzime given to such transfer procedure is Northern blotting. There are some differences in the methodology between Southern and Northern procedures. The first major difference is that RNA is not denatured by alkali because it becomes hydrolysed with such treatment. Instead formaldehyde is used for the purpose. Secondly, RNA does not bind to nitrocellulose unless denatured. Thus, in Northern blotting diazoben lojgrmethyl (DBM) paper is used. This paper binds both RNA and DNA. 

The main procedure of northern blotting is very similar to that of Southern blotting in that both contain steps of sample preparation, gel electrophoresis, transfer from gel to membrane, probe hybridization, and detection. Northern blotting differs from the Southern procedure in the following ways ... 

The electrotransfer of proteins onto (non-specific) binding membranous sheets is named Western hlot in contrast to the transfer of DNA (Southern hlot) and of RNA (Northern hlot). The main advantage of blotting procedures lies in the immobilization and presentation of macromolecules on the surface of a solid planar material. This presentation leads to an easy access of reactants in the opposite to the diffusion-controlled motion of reaction partners within gels or macroporous spheres. 

This procedure is similar in principle to Southern and Northern blots, but it is designed for the transfer of proteins from gels onto nitrocellulose membranes. An electrophoretic technique is often used to speed the transfer by 10-fold, and the apparatus used for such Western transfers is shown in Figure 9.15. The rapid electrophoretic process ensures that complete transfer occurs with minimal diffusional zone broadening. 

For transfer to diazotized membranes (Wahl et al., 1987a,b), DNA is fractionated by electrophoresis as for other Southern procedures. However, after electrophoresis, the gel is rinsed with water and soaked in 1 M NaOAc (pH 4.0, 30 min) followed by rinsing in water and soaking in 20 mM NaOAc for 30 min. Transfer to diazotized membranes is as for nitrocellulose except that 20 mM NaOAc is used as a blotting buffer. Since DNA becomes covalently linked, it is not necessary to bake the membranes. Nylon membranes are generally preferred over these membranes for hybridization. 

Some changes have been made to the basic procedure over the years. The original procedure as described by Southern (1) used the capillary action of the transfer buffer to provide the motive force to transfer the DNA from the gel to the membrane. Vacuum blotting was first... 

The gels used for electrophoresis (agarose, polyacrylamide) are often unsuitable for fiuther stu of the resolved substances, whereas the replica (or blot) can be submitted to a variety of analytical procedures For example, extremely small quantities of DNA can be located by hybridization with radioactive RNA, and proteins can be located and identified with antibodies In the original method of Southern, the nitrocellulose membrane is layered on top of the gel (DNA is first denatured by immersion of the gel in buffered ethidium bromide), followed by moist filter paper and then by several layers of dry filter paper. DNA fragments move from the gel by capillarity and are trapped in the nitrocellulose. For the replication of protein separations, zones may be transferred to nitrocellulose, or to diazobenzyloxymethyl (DBM) paper or diazophenylthioether (DPT) paper. Transfers can be performed more rapi y by electrophoresing the zones from the gel into the receiving layer the replica is then called an electioblot or blitz blot. 

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). 

The main procedures of western blotting are similar to those of Southern and northern blotting, including gel electrophoresis, transfer to a membrane, and probe hybridization. However, there are differences of particular note ... 

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