User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2012/02/01

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Objective

Prepare media for GFP expression next week. Using affinity chromatography, purify the MBP protein with a His-tag, that had previously been attempted to be purified by other members of the class.

Description

Preparation of Media for Expression

  1. Combine 25g LB and 1L of distilled water in a 2.8L flask. (2 flasks)
  2. Combine 0.875g LB, 0.7g agar, and 35mL distilled water in a 250mL flask.
  3. Autoclave the flasks (liquid cycle).
  4. Add 35μL of 100mg/mL ampicillin to the mixture in the 250mL flask when cooled to ~60°C.
  5. Pour the mixture in the 250mL flask into a sterile petri plate.

Filtering the MBP with His-tag

Groups 2-5 in the previous semester attempted to purify the MBP with His-tag, but did not seem to get much protein. Their purification products were saved and stored at 4°C. the groups saved the fractions from their purification in various "peaks" based on the data they observed from the FPLC. Peaks 3 and 4 will be repurified by our group using affinity chromatography. Over the time they were stored it appeared that bacteria began to grow in some of the collections.

  • Using a 60mL syringe, the fractions were pulled into the syringe and then pushed through a 0.2μM filter.
  • The fractions from the different groups were combined, but the peaks were kept separate.

Purifying "Peak 3"

  1. The filtered peak 3 fractions (~100mL) were pumped over the regenerated amylose resin column at 1.5mL/min.
  2. Wash the column with 100mL of column buffer (20mM Tris-HCl (pH 7.4), 200mM NaCl, and 1mM EDTA) at 1.5mL/min.
  3. Elute the protein from the column with 100mL 10mM maltose in column buffer.
    • 10mM maltose(100mL) = 0.36g maltose + column buffer

Streaking out E.coli with MBP

  1. Obtain the glycerol stock (pMXB10) (Stock #115) from the -80°C freezer and store on dry ice.
  2. Using a sterile wooden stick scrape the top of the glycerol stock.
  3. Streak the wooden stick across the petri plate in a zig-zag pattern.
  4. Store the glycerol stock in the -80°C freezer again.