Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

Thin-Layer Chromatography (D. A. Medgyesi)
Thin-Layer Gel Filtration

Thin-layer Gel filtration was proposed by Determann [6], as well as by Johansson and Rymo [11], and proved to be highly useful for microanalysis and rapid comparative analysis of several samples of the material under study. Andrews [1] and later Morris [12] applied thin-layer gel filtration to determine the Molecular Weight of Proteins based on the linear relationship they established between the logarithm of the molecular weight of a given protein and the distance it traveled in the layer of a given carrier over a certain period of time. Accordingly, the molecular weights of unknown Proteins can be estimated by simultaneously performing gel filtration of standard proteins of known molecular weight. When sufficiently sensitive Methods for detecting protein in the carrier layer are available, only a few micrograms of the test material are required to determine the molecular weight. When determining the molecular weight of proteins by thin-layer gel filtration, it should be borne in mind that the mobility of a given protein during Gel Chromatography depends not only on its molecular weight but also on its molecular shape. Therefore, molecular weight determination using gel chromatography is valid only if the molecular shape of the protein under study differs insignificantly from that of the standard proteins used for calibration.

For thin-layer gel filtration, the carrier layer is prepared from the finest gel beads. Among commercially available preparations, "superfine" Sephadex dextran gels or polyacrylamide Bio-Gels of "—400 mesh" are used. In the thin-layer gel chromatography Procedures described in the literature, The Use of Sephadex is generally recommended, but in this case, Bio-Gels are similar to Sephadex.

Gel Swelling occurs under the same conditions and is carried out in the same way as when preparing larger gel particles for Column chromatography. During gel swelling, it is advisable to use a larger volume of solution than the minimum required According to the Water regain capacity, and to remove the excess liquid above the gel before applying it to the plate.

The most suitable carrier layer thickness is 0.5 mm. To apply the carrier onto the plates, the devices described above can be used, which allow obtaining layers of various thicknesses. If the carrier suspension is rolled out with a Glass rod with several turns of electrical tape wrapped around both ends, a sufficiently even layer is also formed. Freshly prepared plates are air-dried for 20–25 min. They can be stored for a relatively long time in a humid chamber, provided that bacterial growth is prevented. In descending thin-layer gel filtration, the plate is placed in the chamber at an angle of 10–15°. As a rule, gel filtration is carried out In aqueous solutions, so the chromatographic chamber can be made of plastic. It consists of a buffer solution trough, a frame supporting the plate at the appropriate angle, and a lid. The buffer solution is supplied to the plate using a filter paper wick. It is convenient to use colored high-molecular-weight substances as markers (for example, fluorescein-labeled ferritin or γ-globulin) that are not retained by the gel particles. Gel filtration is continued until the marker has traveled at least 10 cm from the starting line. After that, the plate is removed from the frame, and the carrier layer is covered with a sheet of filter paper (for example, Schleicher & Schuell 2043 or Whatman 3MM) cut to the size of the plate. Some researchers recommend using a sheet of dry filter paper in this case. In our laboratory, we use moistened and thoroughly squeezed filter paper, as it makes it easier to cover the carrier layer without forming air bubbles under the paper. The paper is then removed (sometimes along with gel particles), dried at a Temperature of about 120°C, and stained with protein-detecting Dyes or Pauly's reagent. Along with other dyes, one can use, for example, Amido Black 10B, Acid Fuchsin, etc. During the washing of the unbound dye, the gel particles separate from the paper, and after drying, it can be used for documentation.

In our laboratory, thin-layer gel filtration on Sephadex G-150 is used to analyze immunoglobulin molecule fragments obtained by Enzymatic Hydrolysis. 5.0 g of Sephadex G-150 is allowed to swell in 150 ml of buffer solution (0.075 M phosphate buffer, pH 7.0, containing 0.075 M NaCl). After 5–6 h of gel filtration, the hydrolysate is separated into an undigested IgG fraction, a divalent fragment fraction (5S), and a monovalent fragment fraction (3.5S), which are clearly distinguishable on the chromatogram (Fig. 47).

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Fig. 47. Thin-layer gel filtration of enzymatically cleaved human IgG on Sephadex G-150.

Spots on the left: Papain hydrolysate obtained without prior oxidation of SH groups (top: undigested protein, bottom: a mixture of Fab and Fc fragments); middle spot: F(ab')₂ fragments obtained by Pepsin Hydrolysis; spot on the right: a mixture of Fab and Fc fragments obtained by papain hydrolysis.

The combined use of thin-layer gel filtration with Electrophoresis or immunodiffusion remains one of the most sensitive methods for protein microanalysis. Hanson et al. [10] developed a two-dimensional Separation method used for protein analysis. In The First stage, proteins are subjected to thin-layer gel filtration on Sephadex G-200 or G-100, and In the second stage, to electrophoresis. They proposed an apparatus in which the chromatographic plate can be mounted at an angle for gel filtration and horizontally for electrophoresis. In the described experiments, 30x30 cm glass plates with a thickness of 1 mm were used, onto which a 0.5 mm thick layer of Sephadex gel was applied. For swelling, Sephadex was left in a 0.05 M veronal buffer solution, pH 8.6. Gel filtration was performed first, followed by electrophoresis in a direction perpendicular to the first for 3 h at a voltage gradient of 10 V/cm. This method has been very successfully applied to the analysis of human Blood sera, CEREBROSPINAL FLUID, and Growth Hormone.

Grant and Everall [8] and Hanson et al. [10] proposed an immuno-gel filtration technique. After gel filtration, the Sephadex located on both sides of the separation zone of the test mixture is removed, and the cleared surface of the plate, as well as the remaining Sephadex layer, is covered with molten 1% Agar cooled to 50°C. After the agar solidifies, troughs are cut into it and filled with the appropriate antiserum. All subsequent steps of the Procedure do not differ from conventional Immunoelectrophoresis [10]. A later Modification of the method [9] allows for quantitative immunochemical Analysis of the fractions separated in the thin layer of Sephadex. Upon completion of gel filtration, the Sephadex layer is covered with an agarose gel plate containing the corresponding Antibodies. As a result of the interaction between the fractionated proteins and the antibodies, precipitation rings are formed, similar to those observed in radial immunodiffusion.

A method has also been described which, in principle, represents a reverse modification of the technique mentioned above [5]. First, an agar plate is prepared, in which gel strips are then cut out and removed, and the vacant space is filled with a Sephadex suspension. Gel filtration is then carried out in the Sephadex layers formed in this manner. The subsequent steps are the same as in immunoelectrophoresis.

References

1. Andrews P., Biochem. J., 91, 222 (1964).

2. Arx E. V., Neher R., J. Chromatogr., 12, 329 (1963).

3. Ballieux R. E., Sehens G., Mul N. A., Protides Biol. Fluids, 14, 527 (1966).

4. Brenner M., Niederwieser A., Pataki G., Experientia, 17, 145 (1961).

5. Carnegie P. R., Pacheco G., Proc. Soc. Exp. Biol., 117, 137 (1964).

6. Determann H., Experientia, 18, 430 (1962).

6a. Gelotte B., Flodin P., Killander J., Arch. Biochem. Biophys., Suppl., No. 1, 319 (1962).

7. Glaesmer R., Buckpaul R., Jung W., Z. Med. Labortech., 6, 175 (1965).

8. Grant G. H., Everall P. H., J. Clin. Path., 18, 654 (1965).

9. Hanson L. A., Holmgren J., Wadsworth C., Int. Arch. Allergy Appl. Immunol., 40, 806—819 (1971).

10. Hanson L. A., Johansson B. G., Rymo L., Clin. Chim. Acta, 14, 391 (1966).

11. Johansson B. G., Rymo L., Acta Chem. Scand., 18, 217 (1964).

12. Morris C. J. O. R., J. Chromatogr., 16, 167 (1964).

13. Nybom N., Physiol. Plantarum, 17, 434 (1964).

14. Ritschard W. J., J. Chromatorg., 16, 327 (1964).

15. Seiler N., Weichmann J., Experientia, 20, 559 (1964).

16. Stahl E., Thin Layer Chromatography. A Laboratory Handbook, Springer, Berlin, 1969.

17. Stegemann H., Lerch B., Anal. Biochem., 9, 417 (1964).

18. Wieland T., Georgppulos D., Biochem. Z., 340, 476 (1964).

19. Woods R. R., Wang R. T., Biochim. Biophys. Acta, 133, 369 (1967).

Recommended reading

Pataki G., Dünnschichtchromatographie in Aminosäure- und Peptidchemie, Walter de Qruyter, Berlin, 1966.

Randerath R., Thin Layer Chromatography, Academic Press, New York, 1965.