Expression testing
Use ~50ml growth per reaction. If your expression is good, you can use less or split the reaction.
If you are growing a 50ml culture from a plate, it is approx 5hrs from inoculation to induction. If you are inoculating from a pre-culture it is approx 2hrs from inoculation to induction. Grow cells in a small conical flask exactly as for a large-scale growth in LB media. Alternatively, grow a whole litre, and spin down 50ml separately for small-scale tests leaving the main pellet for further purification.
Spin down cells in 50ml falcon tubes on max speed for 20mins. Resuspend the pellet in 1.5ml falcon tube in 1ml lysis buffer.
Sonicate for 10s (or 2x10s) using the small tip and a cycle of 1s on, 2s off if possible (not possible with Schofield small-tip sonicator). Spin at top speed in a pre-cooled centrifuge for 20mins.
Prepare 50ul resin per reaction (you'll need to calculate how much slurry this requires from the % mix of resin and buffer in your resin stock). Put the required amount of slurry in an eppendorf tube. Wash in 1ml buffer and spin at 1500 rpm for 1-2 mins. Remove supernatent carefully. If the beads are stuck up the side of the tube, leave the tube to stand on ice for a few mins and they will settle to the bottom.
Add supernatent from lysis to the beads and incubate on rollers at 4oC for 30mins - 2hrs (depending on resin - longer for GST, shorter for Ni-NTA). Spin down tubes to collect beads and remove supernatent. Keep for a gel to show unbound fraction.
Wash beads 3-4 times, each time adding 1ml buffer, vortexing briefly (if not otherwise resuspended), then centrifuging and discarding supernatent.
For expression testing, add equal amount of SDS loading buffer to clean 'dry' beads. Boil, then spin sample down before loading supernatent onto the gel. This gives an even loading between samples and avoids questions of how many beads have been loaded into each well.
For further reactions / pull-downs, use 'dry' beads for the addition of a second protein / further reaction.