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.
Department of Material Science, Faculty of Science, Ehime University, Matsuyama, Japan. yoji@dpc.ehime-u.ac.jp
Y
Y
Shimazaki
S
S
Ohara
T
T
Manabe
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