Affinity Separation and Characterization of IgG Subfragments by Fast Protein Liquid Chromatography with HiTrap_r Protein A Column HiTrap_r Protein A Kolon İçeren Hızlı Protein Sıvı Kromatografisi ile IgG Altbirimlerinin Afinite
نویسندگان
چکیده
Antibadi moleküllerinin proteolitik enzimlerle alt birimlerine parçalanabildiği bilinmektedir. Bu alt birimler, antibadilerin yapısı ve işlevlerinin belirlenmesi çalışmalarında önemli veriler sunmaktadır. Deneysel uygulamalarda ve immünokimyasal teknikler için antibadi alt birimleri, parçalanmamış bütün antibadi moleküllerine göre önemli avantajlar sunmaktadır. Alt birimlerin boyutları daha küçüktür, kimyasal ve fiziksel dirençleri daha yüksektir. Sunulan çalışmada, insan immunoglobulin G parçalanması için papain kullanılmıştır. Elde edilen alt birimler, hızlı protein sıvı kromatografisi (FPLC) sistemi ile ayrılmıştır. Bu amaç için GE Healthcare HiTrap_r Protein A FF kolon kullanılmış, ayırıcılık (R s ) ve teorik plaka sayısı (N) hesaplanmıştır. Kolona tutunan ve tutunmayan alt birimler, FPLC sisteminin Frac 920 fraksiyon toplama birimi ile toplanmış ve enzim-bağlı immünosorbent kit (ELISA) ve sodyum dodesil sülfat-poliakrilamid jel elektroforez (SDS-PAGE) yöntemleri ile karakterize edilmiştir.
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