A rapid and efficient two-step gel electrophoresis method for the purification of major rye grass pollen allergens.
Purified proteins are mandatory for molecular, immunological and cellular studies. However, purification of proteins from complex mixtures requires specialised chromatography methods (i.e., gel filtration, ion exchange, etc.) using fast protein liquid chromatography (FPLC) or high-performance liquid chromatography (HPLC) systems. Such systems are expensive and certain proteins require two or more different steps for sufficient purity and generally result in low recovery. The aim of this study was to develop a rapid, inexpensive and efficient gel-electrophoresis-based protein purification method using basic and readily available laboratory equipment. We have used crude rye grass pollen extract to purify the major allergens Lol p 1 and Lol p 5 as the model protein candidates. Total proteins were resolved on large primary gel and Coomassie Brilliant Blue (CBB)-stained Lol p 1/5 allergens were excised and purified on a secondary "mini"-gel. Purified proteins were extracted from unstained separating gels and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses. Silver-stained SDS-PAGE gels resolved pure proteins (i.e., 875 microg of Lol p 1 recovered from a 8 mg crude starting material) while immunoblot analysis confirmed immunological reactivity of the purified proteins. Such a purification method is rapid, inexpensive, and efficient in generating proteins of sufficient purity for use in monoclonal antibody (mAb) production, protein sequencing and general molecular, immunological, and cellular studies.
Levy
D
D
Department of Allergy, Asthma and Clinical Immunology, The Alfred Hospital, Monash University Medical School, Prahran, Victoria, Australia.
Davies
J
J
O'Hehir
R
R
Suphioglu
C
C
D000485:Allergens / Q000302:isolation & purification*; D000818:Animals; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D008129:Lolium; D010940:Plant Proteins / Q000302:isolation & purification; D011058:Pollen / Q000737:chemistry*
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C042814:Lol p I protein, Lolium perenne; C084941:Lol p V protein, Lolium perenne; D010940:Plant Proteins