Purification of the basic protein avidin using Gradiflow technology
The Gradiflow, a preparative electrophoresis instrument, which separates proteins on the basis of charge or size, was used to purify the basic protein avidin, p I 10, from chicken egg white. Using a charge based separation at pH 9.0, the high p I of avidin and lysozyme (p I 10.7) allows them to be e...
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| Published in: | Protein expression and purification Vol. 26; no. 1; pp. 149 - 152 |
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| Format: | Journal Article |
| Language: | English |
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Elsevier Inc
01-10-2002
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| Abstract | The Gradiflow, a preparative electrophoresis instrument, which separates proteins on the basis of charge or size, was used to purify the basic protein avidin, p
I 10, from chicken egg white. Using a charge based separation at pH 9.0, the high p
I of avidin and lysozyme (p
I 10.7) allows them to be easily separated from remaining egg white proteins, as these are the only positively charged proteins. In a second step at pH 10.2, the negatively charged avidin is separated from the positively charged lysozyme. This sequential two-step protocol was complete within 4.5
h. Enzyme immunoassay of avidin fractions obtained indicated recoveries of 60–65% from one egg white with minimal lysozyme activity detected. |
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| AbstractList | The Gradiflow, a preparative electrophoresis instrument, which separates proteins on the basis of charge or size, was used to purify the basic protein avidin, p
I 10, from chicken egg white. Using a charge based separation at pH 9.0, the high p
I of avidin and lysozyme (p
I 10.7) allows them to be easily separated from remaining egg white proteins, as these are the only positively charged proteins. In a second step at pH 10.2, the negatively charged avidin is separated from the positively charged lysozyme. This sequential two-step protocol was complete within 4.5
h. Enzyme immunoassay of avidin fractions obtained indicated recoveries of 60–65% from one egg white with minimal lysozyme activity detected. The Gradiflow, a preparative electrophoresis instrument, which separates proteins on the basis of charge or size, was used to purify the basic protein avidin, pI 10, from chicken egg white. Using a charge based separation at pH 9.0, the high pI of avidin and lysozyme (pI 10.7) allows them to be easily separated from remaining egg white proteins, as these are the only positively charged proteins. In a second step at pH 10.2, the negatively charged avidin is separated from the positively charged lysozyme. This sequential two-step protocol was complete within 4.5h. Enzyme immunoassay of avidin fractions obtained indicated recoveries of 60-65% from one egg white with minimal lysozyme activity detected. |
| Author | Rothemund, Deborah L Thomas, Theresa M Rylatt, Dennis B |
| Author_xml | – sequence: 1 givenname: Deborah L surname: Rothemund fullname: Rothemund, Deborah L email: drothemund@gradipore.com – sequence: 2 givenname: Theresa M surname: Thomas fullname: Thomas, Theresa M – sequence: 3 givenname: Dennis B surname: Rylatt fullname: Rylatt, Dennis B |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/12356482$$D View this record in MEDLINE/PubMed |
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| Cites_doi | 10.1016/0003-2697(81)90456-5 10.1016/S0021-9673(99)00807-9 10.1016/0006-291X(68)90774-2 10.1042/bj2650301 10.1042/bj0890591 10.1016/0076-6879(86)22152-7 10.1002/elps.1150160117 10.1016/S0065-3233(08)60411-8 |
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| References | Margolis, Corthals, Horvath (BIB3) 1995; 16 Cuatrecasas, Wilcheck (BIB6) 1968; 33 Orr, Henry, Zeheb (BIB7) 1986; 22 Henry, Orr (BIB5) 1981; 114 Melamed, Green (BIB1) 1963; 89 Green (BIB2) 1975; 29 Piskarev, Shuster, Gabibov, Rabinkov (BIB8) 1990; 265 Rylatt, Napoli, Ogle, Gilbert (BIB4) 1999; 865 Piskarev (10.1016/S1046-5928(02)00517-X_BIB8) 1990; 265 Henry (10.1016/S1046-5928(02)00517-X_BIB5) 1981; 114 Cuatrecasas (10.1016/S1046-5928(02)00517-X_BIB6) 1968; 33 Green (10.1016/S1046-5928(02)00517-X_BIB2) 1975; 29 Melamed (10.1016/S1046-5928(02)00517-X_BIB1) 1963; 89 Margolis (10.1016/S1046-5928(02)00517-X_BIB3) 1995; 16 Rylatt (10.1016/S1046-5928(02)00517-X_BIB4) 1999; 865 Orr (10.1016/S1046-5928(02)00517-X_BIB7) 1986; 22 |
| References_xml | – volume: 114 start-page: 92 year: 1981 end-page: 96 ident: BIB5 article-title: The purification of avidin and its derivatives on 2-Iminobiotin-6-aminohexyl–Sephrarose 4B publication-title: Anal. Biochem. contributor: fullname: Orr – volume: 33 start-page: 235 year: 1968 end-page: 239 ident: BIB6 article-title: Single step purification of avidin from egg white by affinity chromatography on biocytin–Sepharose columns publication-title: Biochem. Biophys. Res. Commun. contributor: fullname: Wilcheck – volume: 265 start-page: 301 year: 1990 end-page: 304 ident: BIB8 article-title: A novel preparative method for the isolation of avidin and riboflavin-binding glycoprotein from chicken egg-white by the use of high-performance liquid chromatography publication-title: Biochem. J. contributor: fullname: Rabinkov – volume: 22 start-page: 83 year: 1986 end-page: 87 ident: BIB7 article-title: Purification of avidin and its derivatives on 2-iminobiotin-6-aminohexyl–Sephrarose 4B publication-title: Methods Enzymol. contributor: fullname: Zeheb – volume: 89 start-page: 591 year: 1963 end-page: 599 ident: BIB1 article-title: Avidin. 2. Purification and composition publication-title: Biochem. J. contributor: fullname: Green – volume: 16 start-page: 98 year: 1995 end-page: 100 ident: BIB3 article-title: Preparative reflux electrophoresis publication-title: Electrophoresis contributor: fullname: Horvath – volume: 865 start-page: 133 year: 1999 end-page: 145 ident: BIB4 article-title: Electrophoretic transfer of proteins across polyacrylamide membranes publication-title: J. Chromatogr. contributor: fullname: Gilbert – volume: 29 start-page: 85 year: 1975 end-page: 133 ident: BIB2 article-title: Avidin publication-title: Adv. Protein Chem. contributor: fullname: Green – volume: 114 start-page: 92 year: 1981 ident: 10.1016/S1046-5928(02)00517-X_BIB5 article-title: The purification of avidin and its derivatives on 2-Iminobiotin-6-aminohexyl–Sephrarose 4B publication-title: Anal. Biochem. doi: 10.1016/0003-2697(81)90456-5 contributor: fullname: Henry – volume: 865 start-page: 133 year: 1999 ident: 10.1016/S1046-5928(02)00517-X_BIB4 article-title: Electrophoretic transfer of proteins across polyacrylamide membranes publication-title: J. Chromatogr. doi: 10.1016/S0021-9673(99)00807-9 contributor: fullname: Rylatt – volume: 33 start-page: 235 issue: 2 year: 1968 ident: 10.1016/S1046-5928(02)00517-X_BIB6 article-title: Single step purification of avidin from egg white by affinity chromatography on biocytin–Sepharose columns publication-title: Biochem. Biophys. Res. Commun. doi: 10.1016/0006-291X(68)90774-2 contributor: fullname: Cuatrecasas – volume: 265 start-page: 301 year: 1990 ident: 10.1016/S1046-5928(02)00517-X_BIB8 article-title: A novel preparative method for the isolation of avidin and riboflavin-binding glycoprotein from chicken egg-white by the use of high-performance liquid chromatography publication-title: Biochem. J. doi: 10.1042/bj2650301 contributor: fullname: Piskarev – volume: 89 start-page: 591 year: 1963 ident: 10.1016/S1046-5928(02)00517-X_BIB1 article-title: Avidin. 2. Purification and composition publication-title: Biochem. J. doi: 10.1042/bj0890591 contributor: fullname: Melamed – volume: 22 start-page: 83 year: 1986 ident: 10.1016/S1046-5928(02)00517-X_BIB7 article-title: Purification of avidin and its derivatives on 2-iminobiotin-6-aminohexyl–Sephrarose 4B publication-title: Methods Enzymol. doi: 10.1016/0076-6879(86)22152-7 contributor: fullname: Orr – volume: 16 start-page: 98 year: 1995 ident: 10.1016/S1046-5928(02)00517-X_BIB3 article-title: Preparative reflux electrophoresis publication-title: Electrophoresis doi: 10.1002/elps.1150160117 contributor: fullname: Margolis – volume: 29 start-page: 85 year: 1975 ident: 10.1016/S1046-5928(02)00517-X_BIB2 article-title: Avidin publication-title: Adv. Protein Chem. doi: 10.1016/S0065-3233(08)60411-8 contributor: fullname: Green |
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| SubjectTerms | Animals Avidin - isolation & purification Chickens Egg White Electrophoresis - instrumentation Electrophoresis - methods Hydrogen-Ion Concentration Muramidase - metabolism |
| Title | Purification of the basic protein avidin using Gradiflow technology |
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