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Team:Bielefeld-Germany/Results/S-Layer/Guide/6
From 2011.igem.org
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Bilder: HisTrap, ÄKTA, Säulen | Bilder: HisTrap, ÄKTA, Säulen | ||
| - | [http://lmgtfy.com/?q=his-tag Polyhistidine tags] are used for easy purification. | + | [http://lmgtfy.com/?q=his-tag Polyhistidine tags] are used for easy purification. They bind to Cobalt or Nickel ions which are normally immobilized on chromatography beads or in spin columns. We used a 1 mL HisTrap FF crude by [http://www.gehealthcare.com/ GE Healthcare] in combination with a [http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/aktadesign_platform~akta_primeplus GE Healthcare ÄKTAprime™ plus] FPLC device or just with a syringe which is possible with [these columns http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/Products?OpenDocument&moduleid=165904]. We did not test commercial kits for his-tag purification but perhaps it even works with these. After binding the proteins to the column they were eluted with 50 mM imidazol. This fraction was collected and dialysed against ddH<sub>2</sub>O at 4 °C for 18 h using dialysis tubes by [http://www.carlroth.com/catalogue/catalogue.do;jsessionid=36FBA09A4B8B2D86BA4321073514B5FA?favOid=00000009000256d300020023&act=showBookmark&lang=de-de&market=DE Roth] with 6 kDa cut-off. |
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| + | Bilder: SDS-PAGE, NanoDrop, BCA | ||
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| + | After the dialysis you have purified S-layer fusion protein monomers which are ready to assemble (the so called monomer solution). Check the protein concentration (e.g. BCA assay or NanoDrop) and dilute to 1 mg mL<sup>-1</sup>. Check the purity of your monomer solution by SDS-PAGE as well. | ||
Revision as of 21:38, 27 October 2011
His-tag affinity chromatography
Bilder: HisTrap, ÄKTA, Säulen
Polyhistidine tags are used for easy purification. They bind to Cobalt or Nickel ions which are normally immobilized on chromatography beads or in spin columns. We used a 1 mL HisTrap FF crude by GE Healthcare in combination with a GE Healthcare ÄKTAprime™ plus FPLC device or just with a syringe which is possible with [these columns http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/Products?OpenDocument&moduleid=165904]. We did not test commercial kits for his-tag purification but perhaps it even works with these. After binding the proteins to the column they were eluted with 50 mM imidazol. This fraction was collected and dialysed against ddH2O at 4 °C for 18 h using dialysis tubes by Roth with 6 kDa cut-off.
Bilder: SDS-PAGE, NanoDrop, BCA
After the dialysis you have purified S-layer fusion protein monomers which are ready to assemble (the so called monomer solution). Check the protein concentration (e.g. BCA assay or NanoDrop) and dilute to 1 mg mL-1. Check the purity of your monomer solution by SDS-PAGE as well.
