les_5 - 69 VI.3.2. PROTEIN AFFINITY CHROMATOGRAPHY...

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Unformatted text preview: 69 VI.3.2. PROTEIN AFFINITY CHROMATOGRAPHY VI.3.2.1. Principle A cell extract is passed over a column on which a specific protein is immobilized. Proteins in the cell extract that do not interact with the protein on the column will be present in the flow through, whereas interacting proteins will be retained on the column. After washing, the target molecule is eluted (dissociated and recovered) from the immobilized protein Fig. 7. Protein affinity chromatography VI.3.2.2. Procedure 1. immobilization of the protein to the column/resin: Use pure protein to immobilize onto the column/resin. Proteins are mostly immobilized to the column/resin by affinity-tags, see table 3. Tag fused to gene of interest Binding properties Elution possibilities Glutathione S-transferase (GST) Oligohistidine Maltose binding protein (MBP) HA c-Myc Flag Staphylococcus protein A Glutathione-agarose Ni 2+ or Co 2+ column Amylose resin Anti-HA Ab (Ig) Anti-cMyc Ab Anti-Flag Ab IgG SDS or glutathione 150-300 mM imidazole (competition with oligohistidine) or low pH 10 mM maltose SDS SDS SDS SDS Table 3. Protein tags or fusions used for protein affinity purification 70 Figure 8. GST pull down. Remark: a recombinant GST-fusion protein is often expressed in and purified out of E. coli. Interactions dependent on post-translational modifications such as glycosylation or phosphorylation may not be detected. Some remarks: c Influence of posttranslational modifications : Express proteins in appropriate host cells e.g. SH2 domain interaction with P-Tyr is not detectable in bacteria. c Retention of native structure of the coupled protein. Sometimes coupling can inactivate the protein of interest, also an absence of all subunits of the complex your protein belongs to can give problems. Therefore, if possible, test your protein for activity. 2. Load cell extract onto the column and wash several times Non-specific (e.g. simple ionic) binding interactions can be minimized by adding low levels of detergent or by moderate adjustments to salt concentration in the binding and/or wash buffer. 3. Elution can be done in different ways, dependent on the affinity method that is used (see table 3 and 4) Elution buffer can dissociate binding partners by extremes of pH (low or high), high salt (ionic strength), the use of detergents or chaotropic agents that denature one or both of the molecules, removal of a binding factor or competition with a counter ligand. 71 Condition Buffer pH 100 mM glycine•HCl, pH 2.5-3.0 100 mM citric acid, pH 3.0 50-100 mM triethylamine or triethanolamine, pH 11.5 150 mM ammonium hydroxide, pH 10.5 Ionic strength and/or chaotropic effects 3.5-4.0 M magnesium chloride, pH 7.0 in 10 mM Tris 5 M lithium chloride in 10 mM phosphate buffer, pH 7.2 2.5 M sodium iodide, pH 7.5 0.2-3.0 sodium thiocyanate Denaturing 2-6 M guanidine•HCl 2-8 M urea 1% deoxycholate 1 % SDS Organic 10% dioxane 50% ethylene glycol, pH 8-11.5 (also chaotropic) Competitor >0.1 M counter ligand or analog>0....
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This note was uploaded on 05/28/2010 for the course WE BIBI000000 taught by Professor Johangrooten during the Spring '10 term at Ghent University.

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les_5 - 69 VI.3.2. PROTEIN AFFINITY CHROMATOGRAPHY...

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