sugar experiment

Sugar experiment for FslA solubility

Obtain four ependorff tubes and fill them in the following way:

• Tube #1: negative control
  • 144uL FsLA ITC buffer
  • 6uL purified R351Q FslA protein (25mg/mL)
• Tube #2: positive control
  • 144uL ITC buffer with 25% glycerol
    • note: ITC buffer stock only has 10% glycerol, so add 15% for a total of 25%
  • 6uL purified R351Q FslA protein (25mg/mL)
• Tube #3: 50mM trehalose
  • 7.5uL trehalose sugar
  • 136.5uL ITC buffer
  • 6uL purified R351Q FslA protein (25mg/mL)
• Tube #4: 100mM trehalose
  • 15uL trehalose sugar
  • 129uL ITC buffer
  • 6uL purified R351Q FslA protein (25mg/mL)

Note: the point of this procedure is that the protein concentration is at 1mg/mL and does not need to be diluted when taking measurements. Make sure protein is actually at 1mg/mL before starting this experiment. Might need to change the amounts of protein and buffer accordingly to make measuring the absorbance easier.

Take a zero point (!) before incubating to measure absorbance at 280nm. Then, place the four ependorff tubes in the incushaker at 37C and shaking at around 230rpm. At the 30min, 60min, and 90min points, remove the tubes, centrifuge at 10000xg for about one minute, then take the absorbances using the Nano drop. Place back in incubator and repeat for the rest of the time points.

 

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