User:Steven Moss/Notebook/AU CHEM-570/2012/02/07

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Objective

  • Begin GFP expression, observe purification of peak 3 (MBP with his tag) fractions and begin purification of peak 4 fractions if necessary.

Procedure

GFP Expression Prep

  • create 0.1 M Isopropyl β-D-1-thiogalactopyranoside (IPTG) solution for lac operon expression
  • create Ampilcillin solution at 100 mg/mL of water
  • 35 μL Ampicillin soln and 1 E. coli colony to previously prepared 35 ml LB media (repeat 4 times)
  • make sure IPTG soln is ready for addition in next day
  • incubate overnight at 37°C at 250 rpm

Testing Fraction 3 MBP Purification

  • test each fraction using Bio-Rad Protein Assay using Bradford "by-eye"

Starting Fraction 4 MBP Purification

  • column seemed clean after previous run
  • run peak 4 solns at 0.5 ml/min over amylose resin collecting 5 ml fractions
  • run maltose mobile phase afterwards at 0.5 ml/min. Let run overnight

Data

Testing Fraction 3 MBP Purification

  • it appeared that most of the protein eluted in the initial wash and did not bind to the affinity column because most of the initial spots appeared blue while in the later purification, only 2 of the fractions contained any protein
  • this was considered inconclusive, and the protein that was eluted was not further tested.