Hardy E, Quintana D, Pentón G, Abrahantes MC

Language: English
References: 6
Page: 2301-2305
PDF size: 417.14 Kb.

ABSTRACT

Forcing excised gel fragments through a metal sieve of 32 µm average mesh size contained in the bottom of a 1 mL syringe is a useful method to enhance passive elution of proteins. This extrusion technique works by fragmenting the gels into extremely small pieces, to maximize surface area and minimize the mean distance each protein molecule must diffuse. However, the microgel crusher is not practical when tens of 0.5-1.5-mm-thick gel slices are simultaneously processed for recovering milligram amounts of target protein. The entire procedure is time-consuming, rather tedious, labor-intensive, and requires skills. These disadvantages are also manifested when even a single, thicker (e.g., 6-12 mm) gel slice is processed for protein elution. Here, we propose solving this problem by replacing the microgel crusher with a high-speed homogenizer (Ultraturrax^®, Ika). The applicability and utility of the new procedure was demonstrated in the elution and recovery of milligrams of polyacrylamide gel electrophoresis-separated pertactins (Prn). The overall elution yields were 67.6 % for Prn type 1 (6.5 mg) and 88.5 % for Prn type 2 (8.5 mg total). On average, the recovery from the process was 78 ± 14.8 % protein. As expected, the purified proteins did not show any noticeable variation in migration rate or any visible degradation products, and were detected with an anti-Prn1 monoclonal antibody (PeM19). Consequently, the new protocol will be useful for the milligram-scale isolation of target proteins separated in thick polyacrylamide gels.

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