A simple method for non-denaturing purification of biotin-tagged proteins through competitive elution with free biotin
| Autor: | Audrey Mitchell, Kui Lin, Natasha Funk, Qin Yan, Catherlin Lu, Hao Xiao |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Streptavidin
Proteomics Protein Denaturation Immunoprecipitation Biotin 030204 cardiovascular system & hematology General Biochemistry Genetics and Molecular Biology Antibodies 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Affinity chromatography Carbon-Nitrogen Ligases Bovine serum albumin 030304 developmental biology 0303 health sciences Chromatography biology Elution Escherichia coli Proteins Proteins Serum Albumin Bovine Hydrogen-Ion Concentration Repressor Proteins chemistry Biotinylation biology.protein Biotechnology Avidin Chromatography Liquid |
| Zdroj: | BioTechniques. 68(1) |
| ISSN: | 1940-9818 |
| Popis: | The use of avidin or streptavidin in the purification of biotinylated proteins has been highly restricted due to the harsh and denaturing elution conditions. Here, we use biotinylated bovine serum albumin as a working model to demonstrate a simple and rapid method for biotin-tagged protein purification under non-denaturing conditions. The biotinylated bovine serum albumin is specifically bound to the anti-biotin antibody agarose beads and competitively eluted with free biotin under near-neutral conditions. The optimized elution conditions include using 4 mg/ml biotin (pH 8.5) as the elution buffer and allowing the buffer to incubate with agarose beads for 30 min prior to elution. The elution recovery rate is over 85% without apparent protein denaturation. The method is applicable for both immunoprecipitation and column chromatography. |
| Databáze: | OpenAIRE |
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