Isolation of GFP by Chromatography and PAGE (Production of Cell Lysates (…
Isolation of GFP by Chromatography and PAGE
Production of Cell Lysates
Look at overnight transformed E. coli under UV light
1.7 ml of culture into microcentrifuge tube and spin at max speed for 5 minutes. Discard supernatant in 'bacterial waste'
Resuspend pellets with 250 ul of TE buffer and transfer cell suspensions from both tubes into one new microcentrifuge tube
100 ul of lyosome is added and invert tube 810 times then place in 37 C for 15minutes.
place tube in dry ice until it's contents are frozen by using tongs
Hold tube and thaw it and repeat this process two more times
Place tube in 37 C for 10 minutes and spin at 14,000 rpm for 10 minutes
examine supernatant with UV penlight
Isolate GFP by Column Chomotagraphy
Transfer supernatant to a new microcentrifuge tube, label 'crude extract' and and place in ice.
label 3 test tubes 1-3. place HIC column on 1. Remove top cap and snap bottom cap and allow buffer to drain on tube 1.
add 400 ul of sueprnatant and 400 ul of Binding buffer to a new tube, invert and place in ice.
Add 2 ml of Equilibration buffer to the top . cap the bottom of column and apply 800 ul of preparation to the column. Let supernatant drip down the side of the column wall. xamine with UV light
Transfer column into tube 2 and add 2 ml of wash buffer to column. observe with UV light. Add 1 ml of TE to column. Let column drip onto tube 2.
When GFP comes out move to tube 3. Add more TE and then cap column when finished.
Mix contents in tube 2. transfer 300 ul and set on new microcentrifuge 'fraction 2' and pace on ice...Same idea for tube 3.
Purification of GFP by SDS-PAGE
Obtain, thaw, and mix each sample
place 20 ul of 'crude extract' into a new microcentrifuge and 20 ul of protein loading buffer. Label 'CXB' and store in ice
place 20 ul of fraction 2 into a new microcentrifuge and 20 ul of protein loading buffer. Label '2B' and store in ice. same idea for fraction 3.
Place Marker, 'CXB', '2B', '3B'AT 65 C for 5 minutes then place in ice
Load 6 ul Marker into second well of gel
Load 30 ul of each sample to wells
Run gel for 1 hour at 120 volts
strain and destrain gel before taking a photograph