Small scale fusion protein preparation
Grow 5ml culture o/n in TB with amp.
Add o/n culture to 50ml of TB+amp and grow for 3 hours in 37_C shaker.
Induce culture by adding 20μl of 1M IPTG (final [0.4mM]) and transfer to 25_C shaker for 1hour.
Pellet Bacteria 10min at 3K
Resuspend bacteria in 1ml of NETN+protease inhibitors.
Sonicate 2x for 5-10seconds each time.
Spin out cell debris by spinning in cold microfuge 5 min. at max.
Remove supernatant and store at -70_C.
Binding fusion protein lysate to beads
Remove 400μl of GST beads into eppendorf and spin 15 sec at 8k.
Remove supernatant and wash 2x with NETN.
Resuspend beads in 320μl of NETN (~550μl final volume)
Aliquot 50μl of beads and add up to 200μl of GST lysate. If < 200μl bring final vol. up to 200μl with NETN.
Incubate at 4_C with rocking for 30min.
Wash 3x with NETN and add lysate containing protein of interest.
Incubate 1-2hr at 4_C with rocking.
Wash 3x with appropriate salt buffer.
Boil proteins off in 2xSB for western blotting.
NETN 2xSB
20mM Tris pH8.0 125mM Tris pH6.8
100mM NaCl 20% Glycerol
1mM EDTA 4.1% SDS
0.5% NP-40 2%BME
0.005% Bromphenol Blue
GST Purification Large Scale Protocol
Resuspend 500ml pellet in 10ml of NETN and mix well (Keep at 4_C)
Combine two pellets (20ml) and transfer 20ml of resuspension to 50ml conical
Sonicate 2x with 15 sec pulses
Spin for 20 at 10k in Sorvall at 4_C.
Transfer supernatant into new 50ml tubes.
Add 1ml of washed beads rock for 30min at RT
Pour sample into column and drain
Save flow through and repeat previous step 2x
Wash beads with 10ml of 1xPBS 3x
Add 500μl of Elution buffer and rock for 10min at RT
Drain and collect 500μl of fraction
Repeat 2x more
Elution Buffer (5ml) NETN
20mM Tris pH 8.0
10mM glutathione 15.6mg 100mM NaCl
50mM Tris pH 8.0 125μl 1mM EDTA
H2O 4.9ml 0.5% NP-40
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