Comparative studies on tree pollen allergens. XIII. Partial characterization of the alder (Alnus incana) pollen extract by two-dimensional IEF/SDS-PAG electrophoresis combined with electrophoretic transfer and immunoautoradiography.
Horizontal isoelectric focusing (IEF) and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) combined with electrophoretic transfer (ET) and immunoautoradiography (IAR) were used to characterize the crude alder (Alnus incana) pollen extract (AI crude) and fraction AI 34. IEF resolved AI crude and AI 34 into 34 and 39 distinct protein bands, respectively, when stained with Coomassie brilliant blue. The band patterns were similar and the majority of bands were located in the pH region 4.0-7.0. After ET and IAR, the distribution of antigenic reactivities was similar to that of the protein stain. Allergenic reactivity was detected in the pH region 4.6-5.2 only, where two bands at pH 4.9 demonstrated dominating IgE-binding properties. The 2-D reference maps of AI crude and AI 34 consisted of 40 and 45 protein spots, respectively. The autoradiogram of AI crude demonstrated 35 spots capable of binding rabbit IgG. The main IgE-binding zone was located at pH 4.6-5.2 and 20 kilodaltons with an area of weaker autoradiographic signals at pH 4.6-5.2 and 40 kilodaltons. ET from polyacrylamide gels to nitrocellulose (NC) membranes was quantitative and IAR gave significant radiostaining of spots containing 0.01 and 0.1 microgram of AI crude in the IgG and IgE systems, respectively. For the characterization of the alder pollen extract, horizontal 2-D SDS-PAGE combined with ET and IAR provided a powerful supplement to conventional crossed immuno- and radioimmunoelectrophoretic techniques (CIE/CRIE).
D000818:Animals; D001345:Autoradiography; D006801:Humans; D007158:Immunologic Techniques; D007525:Isoelectric Focusing; D010936:Plant Extracts / Q000276:immunology; D011058:Pollen / Q000276:immunology / Q000302:isolation & purification*; D011817:Rabbits
D010936:Plant Extracts