Studies on externally disposed plasma membrane proteins (original) (raw)
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Analytical Biochemistry, 1974
An improved immunochemical procedure for the quantitative isolation of labeled minor proteins from tissue homogenates is worked out and is applied to the isolation of glucose&-phosphate dehydrogenase from mouse liver. Goat anti-enzyme serum is used as primary reagent, followed by rabbit anti-goat IgG, and not by carrier enzyme as in currently used methods. The resulting immunoprecipitates are analyzed by acrylamide gel electrophoresis, so that only counts in enzyme bands are registered. An equivalent precipitate formed with serum from nonimmunized goat serves as an efficient control for coprecipitation. ' G6PDH-Glucosed-phosphate dehydrogenase. 386
Immunoelectrophoretic quantitation of trace proteins
Journal of Immunological Methods, 1976
A method is suggested for increasing the sensitivity of quantitative immunoelectrophoresis to a level comparable with radioimmunoassay. This is achieved by electrophoretic elution of antigen from 0.1--10 ml samples placed in glass reservoirs on the electrophoresis plate.
Biochimica et biophysica acta, 1981
This report describes a study of the protein constituents of pig spleen lymphocyte plasma membrane, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analyzed under reducing and non-reducing conditions. The electrophoretic patterns of purified plasma membranes, of the endoplasmic reticulum fraction and of a non-ionic detergent (Nonidet P-40) extract of intact lymphocytes are compared. The lymphocytes were ruptured by the nitrogen cavitation method and the plasma membranes were purified by differential centrifugation in sucrose gradients. Plasma membranes were enriched in marker enzymes and appeared, in electron micrographs, as vesicles of various sizes and as fragmented membranes. The protein constituents were resolved into more than 60 bands which appeared, except in the endoplasmic reticulum fraction, as discrete Coomassie-positive bands. Characteristic bands were present in each of the three fractions when samples were not reduced. However, the majority o...
Journal of Immunological Methods, 1984
A simple immunobinding procedure for the detection and molecular characterization of antigens is described. Antigen is adsorbed by immobilized antibodies, and this is followed by radiolabeling with iodine. Both adsorption and radioiodination are carried out in microtitcr wells. After gel electrophoresis and autoradiography the apparent molecular weight of the radiolabeled antigen may be estimated. With this procedure we show that 2 monoclonal antibodies, directed against different determinants, both detect a glycoprotein with an apparent molecular weight of 170,000. By a 2.-site sandwich immunoassay we demonstrate that these antibodies detect the same glycoprotein.
Insertion and turnover of macrophage plasma membrane proteins
Proceedings of the National Academy of Sciences, 1979
The composition, insertion, and turnover of externally disposed proteins on the macrophage plasma membrane were analyzed. Cells labeled with [ S~methionine were incubated with the nonpermeant reagent trinitrobenzene sulfonic acid to introduce the trinitrophenyl moiety on free amino groups of externally oriented membrane proteins. The cells were then incubated with rabbit anti-dinitrophenyl IgG and the immune complexes formed with the trinitrophenyl-proteins were isolated from detergent lysates of the cells by using fixed Staphylococcus aureus as the immunoadsorbent. Proteins isolated by this method were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The interval between the release of newly synthesized proteins from ribosomes and their appearance at the cell surface, where they became accessible to trinitrobenzene sulfonic acid, was studied in pulsechase experiments. The "transit" time of four major membrane glycoproteins (48,000-310,000 Md) ranged from 36 to 55 min and their appearance on the cell surface occurred in a relatively synchronous fashion. The turnover of most proteins of molecular weight above 50,000 was very slow (ti/2> 80 hr) and was rather synchronous. Two exceptions were the 310,000 Mr protein, which was lost with a t1/2 = 21 hr, and a major glycoprotein (Mr 48,000), which exhibited more complex kinetics. Although the overall turnover of surface proteins was biphasic in nature, the rapid phase of protein loss was largely due to low molecular weight species.