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The document outlines steps for culturing and inducing E.coli to produce TNF¦Á, GM-CSF, and MOC proteins between November 10-11. On November 14, the proteins will be isolated from the culture suspension according to a specific purification protocol. On November 17, SDS-PAGE will be used to check production of the proteins in crude and supernatant samples. Depending on whether the proteins are in the pellet or supernatant fractions, the purification protocol will be continued under denaturing or native conditions on November 21 or 24. The purification protocol will be finished on November 28. If needed, the sample will be dialyzed to remove urea starting December 1 and the amount of biologically active TNF¦Á will be measured using a
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- 1. 10-Nov to 11-Nov Culture and induce E.coli cultures (TNF¦Á, GM- CSF, MOC), provided by the HAN Biocentre, with IPTG. 14-Nov Isolate proteins from the culture suspension. (250-500ml) (Refer to protocol ¡®purification of polyhistidine-tagged proteins¡¯, start with protocol 4.2 isolation under native conditions till bottom page 14. ) 17-Nov Check the production of TNF¦Á, GM-CSF, MOC by means of SDS-PAGE. (using the crude cell extract prior to centrifugation and the clear supernatant after centrifugation)(Beforehand, determine how much sample should be applied on the SDS-PAGE gel by Dotblot analysis). Stain the gel with CBB. 21-Nov 24-Nov if protein is in the pellet fraction, refer to protocol ¡®purification of polyhistidine-tagged proteins¡¯, continue with protocol 4.3 isolation under if protein is in the supernatant, finish protocol 4.2 denaturing conditions, page 17,No.2) 28-Nov Finish protocol ¡®purification of polyhistidine-tagged proteins¡¯ 1-Dec Dialyze sample to remove urea. (if necessary) 1-Dec Measure the isolated TNF¦Áprotein by Lowry (Determine the amount of biologically active TNF¦Á)