In total, the pellet weighed 1.75 grams, or about 0.6 grams per 100ml, which is lower than the 0.8 grams we have gotten in the past.
Transfer pellet to one falcon tube.
Resuspend in 5 ml/g (8.75ml) of pellet Bug Buster (1x) by pipetting and gently vortexing.
Put on shaker or rotating mixer for 15 min at RT.
Take first fraction, (F1) of this full cell lysate.
Centrifuge 16000 g 20 min at 4 degrees C
Take second fraction of this supernatant (S1) which should contain soluble proteins, while our desired protein should be in the pellet.
Resuspend in the same volume of 1X Bugbuster 8.75ml as above.
Make sure the pellet is completely resuspended!
Take Fraction at this point (F2) Cell lysate #2
Add DNAse (2 ul) and let rotate 20 min. (This may have been too much DNAse but it should be ok.)
Add dry lysozyme to concentration of 200 ug / ml
Dissolved the lysozyme in water and added the water.
Let incubate on ice 30 minutes, swirl every 5 min.
Take another fraction (F3) here, to see if there is a difference after adding the DNAse and the lysozyme.
Add 6 volumes of 1:10 diluted bugbuster (.1X)#*
6x3.4= 20.4 ml
Can split up into two falcon tubes if necessary.
Centrifuge 16000g 15 min. 4 degrees C to collect inclusion bodies.
Collect supernatant as fraction (S2). Inclusion body should be the pellet.
Resuspend pellet in 1/2 volume of original 0.1X bug buster solution
10.2 ml
Mix to get an even suspension by pipetting and vortexing for several minutes and spin down as in step 9.
Take supernatant fractions for each wash step. (s3)
Repeat step 15-16 one more time.
The BUgBuster manufacturer's protocol calls for two more times, but I the Honeybee paper does not mention multiple wash steps, and it seemed like we were losing a sizable portion of our product with each subsequent wash step.
Resuspend inclusion body pellet in 3% SDS solution and incubate at 60 C in the water bath for 2 hours.