Blotting

 Blotting is a method in which a macromolecule is immobilized on a blotting matrix and subsequently probed with a detectable ligand to determine whether the macromolecule binds that specific ligand. The immobilized macromolecule can be DNA, RNA or protein, in which case one generates DNA blots (Southern blots), RNA blots (Northern blots) (1), or protein blots (Western blots) (2, 3). Blots of lipids have also been produced (4). The macromolecule can be applied to the blotting matrix directly (dot blot), or it can be derived and eluted from an electrophoretic gel (gel blot) or even from a bacterial colony or bacteriophage plaque (colony blot).

 1.Typical Blot Analysis

The most common application of blotting involves a complex mixture of DNA, RNA, or protein to be resolved by using a standard gel electrophoresis procedure, such as agarose gel separation of DNA fragments or RNA or SDS-PAGE of protein samples. After electrophoresis, the gel is dismantled from its cassette or glass plates, etc., and a sheet of an appropriate blotting matrix (eg, a nitrocellulose membrane filter;) is cut to size and applied to the surface of the gel. The transfer of the resolved polynucleotides or peptides is accomplished via a procedure called “blotting”, and the blotted macromolecules adsorb to the surface of the matrix while retaining their relative positions, thus creating a faithful replica of the original electrophoretic pattern. The “blot” thus produced is subsequently incubated with a ligand probe, which might be radioactive for detection via autoradiography or conjugated to an enzyme whose activity is detectable. Extensive washing of the blot removes the excess probe from that which is specifically associated with the immobilized macromolecule and remains bound. Subsequent detection of the retained ligand in the complex formed identifies the relevant bimolecular interaction.

 2.Methods for Blotting

2.1. Dot Blotting 

This is the simplest method of applying a sample to be tested (5, 6). The sample can be an unfractionated polynucleotide or protein mixture in solution. A small volume (typically 2 to 5 µl) is applied directly to the surface of a dry blotting matrix by micropipetting the sample onto the matrix or by using commercial vacuum manifolds that enable the application of larger sample volumes  (e.g., 100 µl to 1 ml) by filtration. Such manifolds often create focused and uniform dots or thin slots of sample, thus leading to the terms “dot blots” or “slot blots”, respectively. Typically, an 8cm × 12 cm piece of blotting matrix contains as many as 96 different dots that correspond to the geometry of a standard 96-well ELISA plate. Once created, the dot blot is processed and probed as any other blot, RNA Blots (Northern Blots), Protein Blots ) Western Blots), and Blot Overlays). The advantages of the dot blot procedures are that they do not require any separation process and thus do not subject the sample to undue chemical modifications that could, for example, denature a protein sample (although some denaturation of protein occurs upon adsorption to the matrix). Moreover, dot blots are simple, cheap and quick. Where quantification is intended, direct dot blotting ensures maximal yields of sample recovery. Obviously, however, chromatography or electrophoresis is necessary to resolve a complex sample to ascribe the signal to a specific component.

2.2. Gel Blots

Gel electrophoresis is routinely employed to resolve macromolecules of DNA, RNA, or protein. Agarose gels and polyacrylamide gels can be blotted, and the common goal is to elute the “bands” efficiently from the gel to be immobilized on the surface of the blotting matrix, so as to generate a faithful replica of the electrophoretic pattern. This can be accomplished in a number of ways: 

2.3.Colony Blots 

At times it is necessary to identify a specific bacterial colony or phage that contains DNA of interest

 1.Diffusion blotting simply relies on the fact that the macromolecules in the gel spontaneously diffuse out of the gel (7). Consequently, blots are produced when a blotting matrix is simply applied to one or both sides of the gel. This approach is usually time-consuming (24 to 72 h) and of low efficiency, but it produces two equal copies simultaneously.

2. Convection blotting (also called capillary transfer) is the process of eluting the resolved macromolecules by mass flow of buffer through the gel. This is the original procedure introduced by Edwin Southern for transferring DNA restriction fragments from agarose gels onto nitrocellulose membrane filters (thus the term Southern blotting) (8). In this method, the gel is placed on top of a paper wick, which draws buffer from a reservoir. The gel is covered with a piece of blotting matrix that, in turn, is covered by a stack of paper towels or absorbent paper and a weight that ensures uniform pressure over the surface of the gel. The stack of paper towels draws a continuous flow of buffer vectorially through the gel, and with it the polynucleotides or peptides, which are eluted and deposited on the surface of the blotting matrix (9).

3. Vacuum blotting is an elaboration of blotting by convection in which the flow is accelerated by employing negative pressure (i.e., suction). Positive pressure has also been used to enhance blotting.

4.Electroblotting is achieved by applying an electric field so as to elute the proteins or polynucleotides from their corresponding gels by electrophoresis. This technique has been used primarily for proteins because binding DNA and RNA to nitrocellulose requires high salt conditions that are incompatible with electroblotting. Electroblotting of nucleic acids is possible, however, with alternative blotting matrices, such as nylon membranes. A variety of commercial apparatus equipment and home-made systems are available, including those that generate gradient electric fields and provide different elution efficiencies to compensate for differences in the molecular mass of the polymers to be eluted (10, 11). Electroblotting is performed by using tank systems, which require 2 to 4 liters of transfer buffer, or semidry blotting systems, which conserve buffer and are flexible because different buffers can be employed for the anode and cathode (12).

5.Direct blotting is an elaboration of electroblotting in which a conveyor belt passes the blotting matrix by the exposed bottom of the polyacrylamide gel during electrophoresis. As the proteins or DNA fragments reach the bottom of the gel, they are deposited directly onto the slowly moving blotting matrix and generate a blot. The resolution of the bands is modulated by regulating the conveyor belt speed (13).

2.3. Colony Blots 

At times it is necessary to identify a specific bacterial colony or phage that contains DNA of interest or expresses a particular protein. Libraries of recombinant DNA-containing bacteria or lambda phage expression libraries are used to produce colony or plaque blots to be processed and screened like any other blot. The library of bacteria or phage is plated on agar after suitable incubation, and replicas are produced by simply placing a sheet of blotting matrix, such as nitrocellulose membrane or nylon membrane, onto the colony- or plaque-containing surface. The matrices pick up sufficient material from each colony or plaque at precisely the same relative position corresponding to that on the original agar plate. Then the colony blots are processed, for example, by standard DNA filter hybridization, immunoblotting, or ligand blotting (14, 15) (Fig. 1). 

Figure 1. Bungarotoxin overlay of bacterial colonies. Escherichia coli were transformed with a pATH2 expression vector containing a DNA insert coding for a fragment of the a subunit of the nicotinic acetylcholine receptor. This fragment is responsible for the receptor's binding of the neurotoxin a-bungarotoxin. The bacteria were plated to a density of approximately 200 colonies per agar plate (a) and expression was induced. A replica of the plate was produced by using a nitrocellulose filter disc, which was processed for overlay with radio-iodinated a-bungarotoxin. The filter was subsequently washed and autoradiographed, illustrating those colonies that contain the relevant DNA fragment (b).

References

1.J. Meinkoth and G. Wahl (1984) Anal. Biochem. 138, 267–284. 

2.J. M. Gershoni and G. E. Palade (1983) Anal. Biochem. 131, 1–15. 

3.H. Towbin and J. Gordon (1984) J. Immunol. Methods 72, 313–340. 

4.T. Taki, S. Handa, and D. Ishikawa (1994) Anal. Biochem. 221, 312–316. 

5.J. M. Gershoni (1988) Methods Biochem. Anal. 33, 1–58. 

6.L. G. Davis, M. D. Dibner, and J. F. Battey (1986) Basic Methods in Molecular Biology, Elsevier, New York, pp. 147–149. 

7.B. Bowen, J. Steinberg, U. K. Laemmli, and H. Weintraub (1980) Nucleic Acids Res. 8, 1–20. 

8.E. M. Southern (1975) J. Mol. Biol. 98, 503–517. 

9.Ref. 6 pp. 62–65. 

10. M. Bittner, P. Kupferer, and C. F. Morris (1980) Anal. Biochem. 102, 459–471. 

11. J. M. Gershoni, F. E. Davis, and G. E. Palade (1985) Anal. Biochem. 144, 32–40. 

12. G. Jacobson (1994) In Protein Blotting: A Practical Approach (B. S. Dunbar, ed.), IRL Press, Oxford, UK, pp. 53–72. 

13. S. Beck (1993) Methods Mol. Biol. 23, 219–223. 

14. Ref. 6 pp. 185–189.

15. J. M. Gershoni (1987) Proc. Natl. Acad. Sci USA 84, 4318–4321.