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Protocols and tips in protein purification
How to purify protein in one day. by S.E.Sedelnikova
Protein purification is a first step of almost all in vitro protein studies. High quality of purified protein could be critical in respect to their activity and crystallization ability. Almost all proteins lose their activity during any manipulations. Even stored at 4°C protein can lose activity and crystallization ability. That is why it is important to purify protein as quick as possible. This guide gives information how to purify protein in one day.
1. Introduction
Introduction
2. General sequence of protein purification procedures
Preparation of equipment and reagents
Stock solutions preparation
Preparation of the chromatographic columns
Preparation of crude extract - cell free extract or soluble proteins fraction
Pre-chromatographic steps
Chromatographic steps
3. "Common sense" strategy in protein purification
General principles and tips in common sense strategy
Algorithm for development of purification protocol for soluble over expressed protein
Brief scheme of purification of soluble protein
Purification of DNA-binding proteins
Timing for soluble protein purification procedure
4. Protocols
Preparation of the stock solutions
Quick and effective cell disruption and preparation of the crude extract
Protamin sulphate treatment
Analytical ammonium sulphate cut (AM cut)
Preparative ammonium sulphate cut
Precipitation of proteins by ammonium sulphate
Recovering of protein from the ammonium sulphate precipitate
Preparation of samples for analysis of solubility of expression
Bio-Rad protein assay - Sveta's easy protocol
Protocol for accurate determination of concentration of pure protein
SDS PAGE - Sveta's easy protocol
5. Charts and Tables
Ammonium sulphate -Sugar refractometer
NaCl-Sugar refractometer
Calibration plot for Hi-load Superdex 200 column
Ammonium sulphate table
Typical UV spectrum of tryptophan containing protein
Typical UV spectrum of tyrosine containing protein
6. Appendixes
Determination of protein concentration
How to concentrate proteins
How to store proteins
Protein crystallography
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