Measurement of low level membrane proteins using bicinchoninic acid: modified procedures to eliminate interfering substances.
Accurate determination of low levels of protein in samples containing large amounts of interfering substances is rather difficult. Precipitation-bicinchoninic acid (BCA) assay as reported can concentrate and partially decontaminate the protein by adding sodium deoxycholate (DOC) and trichloroacetic acid (TCA) to the sample. Yet, this procedure alone has been insufficient for analysis of highly contaminated samples. In this report, we describe an improved method, the DOC-TCA-washing-BCA method, which is composed of the DOC-TCA precipitation and a subsequent aqueous washing that eliminates many interfering substances. The protein concentrations in samples containing even large amounts of interfering substances (e.g. various sugars and some detergents) were well quantitated by this method. The modifications described here have enabled us to perform rapid and efficient removal of many interfering substances that are commonly used in protein purification, and to allow proteins to be detected above 0.05 microgram and from a solution above 0.5 microgram/ml.
Luo
Shiwen
S
Department of Pharmacology, Faculty of Medicine, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan.
Takano
Masako
M
Takasaki
Yozo
Y
Asakawa
Takeo
T
D003840:Deoxycholic Acid; D003902:Detergents; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008832:Microchemistry / Q000379:methods; D008970:Molecular Weight; D011804:Quinolines; D014238:Trichloroacetic Acid
D003902:Detergents; D008565:Membrane Proteins; D011804:Quinolines; D003840:Deoxycholic Acid; D014238:Trichloroacetic Acid; C047117:bicinchoninic acid