Protein Expression.
Goals
- Induce Protein expression.
- make the binding buffer and the elution buffer
- Harvest the cells that were grown.
Procedure
- This morning at 8 am, the starter cultures were centrifuged at 4500 rpm for 15 minutes and then resuspended the cell pellets in 4mL of fresh LB.
- Inoculate the expression culture media by dividing the resuspended cells among the Fernbach flasks.
- Incubate the expression cultures at 37°C and 160 rpm until the absorbance of the media at 600nm = 0.6.
- Add 1mL of 0.4M IPTG to each flask in order to induce protein expression
- Continue shaking for 3-4 hours
- Add 30 mL of the binding buffer into each flask
- Harvest the cells by centrifuging at 4500rpm for 15 minutes
- collect the cells and place in -80°C freezer
Calculations
- Binding Buffer: 1 liter total solution at pH 7.4.
a. Theoretical calculations
- 20mM Tris= .020M/L Tris x1 L= .020 mols x 121.14 g/mol = 2.4228 g Tris.
- similar calculations were done for all compounds added into the buffer solution.
- .5M NaCl= 29.2195 g NaCl
- 30mM Imidazole= 2.0424 g Imidazole
- Elution Buffer: 500 mL total solution at pH 7.4
a. Theoretical calculations
- 20mM Tris= .020 × .500= .01 mols × 121.14/ 1mol= 1.2114 g Tris
- similar calculations/stoichiometry was done for all compounded added into the buffer solution.
- .5M NaCl= 14.609 g NaCl
- .5 M imidazole= 17.019 g imidazole.
Conclusions
- sample 1 looked a little different when the samples were looked at this morning prior to centrifuging. It was a different shade of yellow in the erlenmeyer flask.
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