Protein spot recognition on the non-denaturing and denaturing two-dimensional electrophoresis patterns using in situ immunosubtraction via Protein A agarose and antibodies.
Specific proteins in small amounts of human plasma were subtracted from patterns of non-denaturing two-dimensional electrophoresis (non-denaturing 2-DE) by layering a 7 microl aliquot of Protein A agarose-antibody complex on the top of an isoelectric focusing (IEF) gel before the loading of a plasma sample. The Protein A agarose suspension was recovered after non-denaturing IEF and was mixed with 8 M urea-5% 2-mercaptoethanol-1% NP-40 to extract the antibody and the specific plasma protein from Protein A agarose. The extract was then subjected to denaturing two-dimensional electrophoresis (denaturing 2-DE) and the location of the specific polypeptide was determined. The technique can be applied to the extraction and analysis of proteins in small amounts of samples.
Shimazaki
Y
Y
Department of Material Science, Faculty of Science, Ehime University, Matsuyama, Japan. yoji@dpc.ehime-u.ac.jp
Ohara
S
S
Manabe
T
T
D000906:Antibodies; D001798:Blood Proteins / Q000737:chemistry* / Q000276:immunology*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D007525:Isoelectric Focusing; D011489:Protein Denaturation; D012685:Sepharose / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000276:immunology*
D000906:Antibodies; D001798:Blood Proteins; D013205:Staphylococcal Protein A; D012685:Sepharose