GFP protein purification lab
What are we trying to do
• 7.3– Use E.coli that contains pGLO plasmid w/ GFP
gene to express GFP protein
2ml of
culture
What are we trying to do
• 7.3– Use Lysozyme enzyme break open bacterial cells to
release GFP protein (TTh)
– Spin down cellular debris, GFP protein now in
supernatant. Move GFP containing supernatant to
new MCT. Add high salt concentration binding buffer
(decreases solubility of hydrophobic proteins like GFP
(MWF)
What are we trying to do
• 7.3– Use Hydrophobic interaction
column to elute proteins
based on hydrophobicity
• GFP is a relatively Hydrophobic
protein based on its amino
acids. Therefor it holds readily
to the hydrophobic resin beds
in a high salt concentration, but
when the salt concentration is
reduced the GFP goes from
adhering to the beads to being
in solution…and dripping out of
the HIC column
What are we trying to do
• 7.3- extension
… using SDS- page gel to
analyze how purified the GFP
protein became by using HIC
column.
– Protein Standards…by
Kilodalton…why not by
number of amino acids?
– If we want to determine how
purified the GFP was after
7.3, what do we also need to
run in the gel?
• hint: we did not make a lysate
of untransformed E.coli
Making a semi log curve
mass
Distance
Basics of procedure
• 1protein:1 Laemmli buffer
– Compensates for R group variability…Laemmli
buffer makes makes whole protein negatively
charged
• How? SDS (sodium dodecil sulfate) makes protein
negative
Basics of procedure
• 1protein:1 Laemmli buffer
– Compensates for R group variability…Laemmli buffer
makes makes whole protein negatively charged
• How? SDS (sodium dodecil sulfate) makes protein negative
– Run 10 microliters in SDS-page gel in a vertical
Electrophoresis system w/ 1x TGS (tris Glycine SDS*)
*soapy
• 200V
• 30 min
– Stain 1hr Biosafe Coomassie (protein Gel stain)
– Destain overnight in dwater- on rocker
Mini-Protean Tetra cell