Blotting Matrices

 Blotting is the process of transferring macromolecules from electrophoretic gels to immobilizing matrices, called blotting matrices. The variety of matrices available for blotting is quite diverse, and the characteristics of each directly affects the ultimate result and quality in different blot analyses [see Blotting, Southern Blots (DNA Blots), Protein Blots (Western Blots), RNA Blots (Northern Blots), and Blot Overlays]. Generally, two kinds of blot matrices are used: (1) chemically modified paper filters and (2) microporous membrane filters. Although filters are used, blotting depends on chemical adsorption of transferred molecules to the filter material itself, rather than filtration perse, where separation is achieved by size exclusion. Thus porosity is less important than the chemical composition, density, and thickness of the material. After binding of the desired ligand, normally the remaining binding sites on the matrix are blocked with a neutral compound, known as a quencher or blocking reagent.

 1.Paper Filters

Initially, it was thought that transferred molecules would be best suited for blotting if they were covalently bound to the blotting matrix. This led to chemically modified paper filters that contained active moieties to bind DNA, RNA, and protein covalently. For example, cyanogen bromide (CNBr)-activated paper was produced (1), as was diazobenzyloxymethyl (DBM) paper, which was the most popular filter of this type (2). These filters covalently immobilize the blotted macromolecules, but they are cumbersome to handle, have the fibrous texture characteristic of blotting paper, and therefore are rarely used today.

 2.Membrane Filters

 Microporous membrane filters have become the matrices of choice for blotting. These materials are thin films of synthetic polymers with very fine, uniform surfaces. Filters with average porosities of 0.2 to 0.45 µm are extremely suitable for all types of blotting. A variety of materials exist, and each offers unique advantages.

2.1.Nitrocellulose 

Nitrocellulose is by far the most commonly used blotting matrix. It binds DNA, RNA, and protein reasonably well, although the mechanism is not clear (3, 4). Hydrophobic interactions definitely play a role, and the salt conditions are critical, particularly for the adsorption of RNA. Nitrocellulose is often produced as a mixed ester with cellulose acetate, which reduces to some extent its binding capacity for proteins. The advantages of nitrocellulose are that it binds protein well and affords very good signal-to-background ratios in Western blotting assays. It allows staining of the immobilized protein patterns with such dyes as Ponceau S and Amido black. It becomes brittle, however, after baking at 80°C (typical for Southern and Northern blotting), which reduces the repeated use of these filters. Furthermore, nitrocellulose is less resistant to various organic solvents, such as methanol (5).

2.2. Nylon Membranes 

Nylon membranes were introduced initially as an alternative to nitrocellulose for protein blotting (5), and subsequently they were applied in RNA and DNA transfers (4). These membranes are usually derivatives of nylon 66 and often are modified with positive charges. They have proven exceptional binders of DNA, RNA, and protein, but at times this presents some difficulty by producing high backgrounds, particularly in protein blotting. These filters are mechanically stable and thus can be reprobed numerous times without lossing band definition or sensitivity.

2.3. Polyvinyl Difluoride 

Polyvinyl difluoride (PVDF) membrane filters have a special application in blotting where subsequent chemical manipulation of the immobilized macromolecule is desired. A case in point is the use of these membranes in protein blots, where the individual bands are excised out of the blot and subjected to Edman Degradation for N-terminal amino acid sequencing of the resolved and blotted polypeptide chains (6, 7).

References

1.L. Clarke, R. Hitzman, and J. Carbon (1979) Methods Enzymol. 68, 436–442. 

2.J. C. Alwine, D. J. Kemp, and G. R. Stark (1977) Proc. Natl. Acad. Sci. USA 74, 5350–5354. 

3.A. De Maio (1994) In Protein Blotting: A Practical Approach (B. S. Dunbar, ed.), IRL Press, Oxford, UK, pp. 11–32. 

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

5.J. M. Gershoni and G. E. Palade (1982) Anal. Biochem. 124, 396–405. 

6.M. A. Mansfield (1994) In Protein Blotting: A Practical Approach (B. S. Dunbar, ed.), IRL Press, Oxford, UK, pp. 33–52. 

7. C. Eckerskorn and F. Lottspeich (1993) Electrophoresis 14, 831–838.