Simultaneous analysis of microheterogeneity of immunoglobulins and serum protein fraction using high-voltage isoelectric focusing on six cellulose acetate membranes

Abstract

A systematic detection method for the single performance of cellulose acetate (CA) membrane isoelectric focusing to detect six different types of information on protein abnormalities was developed. High‐voltage isoelectric focusing was carried out on six layers of CA membrane using a thermoelectric cooling apparatus. After electrophoresis, the proteins on the top, the third, the fourth, the fifth, and the bottom CA membrane were transferred to a polyvinylidene difluoride (PVDF) membrane by a simple contact printing procedure to detect IgM, κ‐chain, λ‐chain, IgA, and IgG, respectively. Each PVDF membrane revealed the microheterogeneity of these immunoglobulins using specified anti‐serum and enzyme immunostaining. The second CA membrane was stained with Coomassie brilliant blue G250 to detect serum protein patterns. All stained membranes showed clear electrophoretic patterns of immunoglobulin microheterogeneity. By our method, immunoglobulin abnormalities in serum could be screened out using six different types of information obtained simultaneously. J. Clin. Lab. Anal. 11:220–224, 1997. © 1997 Wiley‐Liss, Inc.

Bio-Control of <i>Alternaria alternata</i> during Banana Storage by Purified AFP Using Isoelectric Focusing Technique

“…However, the AFP was not purified from A. giganteus using the isoelectric focusing so far. Meanwhile, many of antifungal protein have been isolated and purified by using the isoelectric focusing system [9]- [11]. The established purification protocol of AFP could be efficient and valuable in AFP purification and yield.…”

“…It is based on the principle that the protein placed in a pH gradient within an electric field will migrate toward either the cathode or anode until it reaches a pH at which there is no net charge on the protein molecule [9]- [11].…”

“…Most recently 16.22 mg•AFP•l −1 were purified using Cellulose phosphate (Cph) as cation exchange chromatography and Sephadex G-50 with incubation the culture at 28˚C for about 100 hrs followed by an additional incubation period for 17 -20 hrs at 37˚C [8]. A few studies were established for purification different antifungal peptides applying the isoelectric focusing procedure [9]- [11], where no one used this technique for purification of AFP from A. gigenteus so far.…”

Acceleration and Improvement of Protein Identification by Mass Spectrometry

“…These parameters are directly linked to the adsorption capacity (Table 2) . Other polymeric based membranes have been used for specific purposes, such as cellulose acetate (Iijima, Shiba, Inoue, Yoshida, & Kimura, 1997), Nylon (mainly used for nucleic acid transfer), polypropylene, or polyethylene (Baker, Dunn, & Yacoub, 1991;. Before the development of polymeric membranes, blotting processes were conducted with derivatised glass fibre tissues and they were mostly used for the Edman sequencing approach.…”

“…A chemical modification of CBB R250 provides the colloidal Coomassie stain, also called Coomassie G250. The major advantage of this stain compared to CBB R250 (Iijima et al, 1997) is its high sensitivity. No direct investigations have been conducted to compare both stains on membranes, but it has been clearly demonstrated in gel based staining .…”