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Unformatted text preview: MIT OpenCourseWare http://ocw.mit.edu 5.36 Biochemistry Laboratory Spring 2009 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms . ________________________________________________________________________________ ________________________________________________________________________________ 5.36 Lecture Summary #3 Thursday, February 12, 2009 CI-M workshop on Feb. 19 th : Bring two hard copies of your mini-review outline and an annotated bibliography w/ at least 3 primary research reports (from 2005 or later). HHMI Summer Research Fellowship in Chemical Biology: http://web.mit.edu/cld/hhmi/ Next Laboratory Session: #4 Topics: Affinity Tags for Protein Purification I. Overview of protein expression and general protein purification strategies II. Affinity tags for protein isolation A. Characteristics of tags B. Common affinity tags (GST, FLAG, and His) C. Cleavage of affinity tags III. SDS gel analysis of purified proteins Please note that we will complete our discussion of Quickchange DNA mutagenesis (continued from Lecture #2 notes) in our March 5 th lecture, prior to lab sessions #9-11. I. PROTEIN EXPRESSION AND GENERAL PURIFICATION STRATEGIES Consider our progress so far for H396P Abl(229-511) expression and isolation: Prior to Session 2, BL21(DE3) expression cells co-transformed with 1) an H396P Abl-encoding ______-resistant vector and 2) a YopH Tyr _______________-encoding _______-resistant vector were spread onto a LB-agar plate with antibiotics for colony selection. kan/strep plate Pick a single colony spin down and freeze cell pellet lyse cells and isolate H396 Abl protein grow up overnight starter culture 500-mL culture induce protein overexpression Successful Abl kinase domain expression in _____________ requires co-expression with a phosphatase! (Seeliger, M.A. et al. Protein Sci . 14, 3135-3139 (2005)) Prior to 2005, expression of active Abl kinase domain was carried out in insect cells. Insect cells yield _______gram quantities of protein, but are time consuming and _______________ to maintain. in bacteria with very low yields (______grams). While biochemical studies can be carried out with tiny amounts of protein, milligram quantities are required ________________________________________________________________________________ ________________________________________________________________________________ 5.36 Lecture Summary #3 Thursday, February 12, 2009 CI-M workshop on Feb. 19 th : Bring two hard copies of your mini-review outline and an annotated bibliography w/ at least 3 primary research reports (from 2005 or later). HHMI Summer Research Fellowship in Chemical Biology: http://web.mit.edu/cld/hhmi/ Next Laboratory Session: #4 Topics: Affinity Tags for Protein Purification I. Overview of protein expression and general protein purification strategies II. Affinity tags for protein isolation A. Characteristics of tags B. Common affinity tags (GST, FLAG, and His) C. Cleavage of affinity tags...
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This note was uploaded on 12/16/2011 for the course BIOLOGY 101 taught by Professor Mr.wallace during the Fall '11 term at Montgomery College.

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536lecntwtbnk_3 11.45.23 PM - MIT OpenCourseWare...

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