GFP protein purification lab
description
Transcript of GFP protein purification lab
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GFP protein purification lab
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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
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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)
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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
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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
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Making a semi log curve
Distance
mass
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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
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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