Prepare column: clean & regenerate & rinse & store in binding buffer

Grow bacteria in rich broth: LB supplemented with 2g/L glucose
IPTG 0.3mM@2-5 hours
Resuspend Bacteria pellet 25ml/L cells in column buffer (pH7.5, 20mM HEPES, EDTA 1mM, beta-mercaptoethanol 10mM, 200mM NaCl, 0.01% azide) supplemented with lysozyme 1mg/ml

freeze >3 hrs

thaw in cold water

Spin at 16,000xg for 30 min to remove debris

add supernatant of lysate to a final volume ~150mL with column buffer
add lysate supernatant to column, let drip by gravity

wash with column buffer with 15X resin bed volumes, repeat 5 times

elute with 5-10 bed volume of column buffer containing 10mM maltose

GST column
Prepare column: clean & regenerate & rinse & store in binding buffer

binding bacterial lysate supernatant overnight in big tube/flask with agitation

pour resin into an empty column

wash A neutral pH7.5, 20mM HEPES, EDTA 1mM, EGTA 1mM, DTT 5mM, 100mM NaCl, azide 0.01%
wash B neutral pH7.5, 20mM HEPES, DTT 1mM, 100mM NaCl, azide 0.01%
Elute with 5-10 bed volmes of pH8.0 reduced glutathione 10mM, 20mM HEPES, DTT 1mM, 100mM NaCl, azide 0.01%

Nickel column (for 6-His protein)

Prepare column: strip with 20mM EDTA, wash with H2O, regenerate with 50mM nickel chloride, wash extensively with H2O

Grow bacteria in LB
IPTG 0.5mM@3 hours
Resuspend Bacteria pellet 25ml/L cells in column buffer (20mM HEPES, 20mM NaCl, azide 0.01%) supplemented with lysozyme 1mg/ml
binding neutral pH7.5, 20mM HEPES, 20mM NaCl, azide 0.01%
wash neutral pH7.5, 20mM HEPES, 150mM NaCl, 20mM imidazole, azide 0.01%
Elute neutral pH7.5, 20mM HEPES, 150mM NaCl, 500mM imidazole, azide 0.01%