Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Glass

Glassware, particularly tubes used for Hydrolysis, must be cleaned by overnight Treatment with a hot sulfuric acid—nitric acid mixture (3:1)*, rinsed thoroughly, and dried vertically in a forced-air oven at 110 °C. Chromic acid should not be used for cleaning glassware due to the potential formation of oxidation products that may remain On the surface. Before using tubes or vials, they should be heated at 550—625° for several hours (placed in a glass container covered with aluminum foil) to destroy any trace amino acid impurities; following this treatment, the glassware must be stored in a sealed, dust-proof container.

When working with microgram quantities of a substance, losses due to adsorption onto vessel walls become significant. These can be minimized by treating the glass surface or suitably modifying the solvent. In this regard, polypropylene or polycarbonate tubes are preferable to glass ones. Glass tubes can be treated with a silylating reagent, such as a 2% solution of dimethyldichlorosilane in carbon tetrachloride [137] or a 5% solution of trimethylchlorosilane in toluene. After soaking for 5 min in one of these solutions, the excess solvent is allowed to drain, the glassware is rinsed with acetone, and dried vertically in an oven at 110 °C.

Glass plates for Polyacrylamide gel Electrophoresis are also silanized [274, 309]. To do this, they are washed with a detergent, Water, and acetone, and then either immersed in or sprayed with a 0.2% solution of Polyfix D (Desaga) in an ethanol—water mixture (1:1). Following this treatment, the plates are dried under an infrared lamp at a distance of ~20 cm (~60 °C); strict adherence to these drying conditions is crucial. Silanized plates can be stored for several weeks.

Glassware can also be treated first with 3-aminopropyltrimethoxysilane and then with glutaraldehyde, followed by The addition of bovine serum albumin (BSA) and reduction with sodium borohydride, or treated with a 1% (w/v) solution of polyethylene glycol 20,000 in deionized water and dried at 110 °C [5].

However, treating glass and plastic surfaces with various Reagents can lead to contamination; therefore, modifying the solvent is a better approach to reducing adsorption losses of Proteins. To this end, the addition of glycerol (up to 50% concentration) and Triton X-100 (up to 0.2 mmol) to the solvent has been successfully employed [356]. When working in plastic containers, either glycerol or Triton X-100 can be used for both low- and high-ionic-strength solutions, whereas for glass vessels, glycerol is recommended for low-ionic-strength solutions and Triton X-100 for high-ionic-strength solutions. It should be noted that the adsorption capacities of different proteins vary considerably.

* Unless otherwise specified, solvent ratios in mixtures are given by volume. — Trans.