Team:UCLA/Notebook/Honeybee Silk/7 August 2015

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8/7

1. Record the weight of the pellets.
   • 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.
2. Transfer pellet to one falcon tube.
3. Resuspend in 5 ml/g (8.75ml) of pellet Bug Buster (1x) by pipetting and gently vortexing.
4. Put on shaker or rotating mixer for 15 min at RT.
   • Take first fraction, (F1) of this full cell lysate.
5. 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.
6. 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
7. Add DNAse (1 ul) and let rotate 20 min. (This may have been too much DNAse but it should be ok.)
8. 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.
9. Take another fraction (F3) here, to see if there is a difference after adding the DNAse and the lysozyme.
10. Add 6 volumes of 1:10 diluted bugbuster (.1X)
    • Can split up into two falcon tubes if necessary.
    • Vortex for 1 minute
11. Centrifuge 16000g 15 min. 4 degrees C to collect inclusion bodies.
12. Collect supernatant as fraction (S2). Inclusion body should be the pellet.
13. Resuspend inclusion body pellet in 3% SDS solution and incubate at 60 C in the water bath for 2 hours.
    • Used 10 ml of SDS. (Final)
14. Store at 4C for further processing and analysis.

Fig. 1 Expected size of product is 40 kDa (lane 6 and 7)). Lane 8 is BSA protein positive control, and lane 1 is Biorad dual color ladder. From lane 1-8 S1,F2,F3,S2,Final product 8/7, Final Product 7/29,BSA positive control, (see table)

Ladder (lane 1)	Bio Rad dual color
S1 (lane 2)	Supernatant after spinning down full cell lysate
F2 (lane 3)	Full cell lysate #2 after resuspending pellet in Bugbuster
F3 (lane 4)	Full cell lysate #3 after adding lysozyme and DNase
S2 (lane 5)	Supernatant after first wash step.
Final product (lane 6)	Purified inclusion body in SDS (8/7)
Final Product (lane 7)	Purified inclusion body in SDS (7/29)
Positive Control (lane 8)	BSA positive control

• You can see that the silk from 7/29 (lane 7) resembles the most recent silk purfication product (lane 6).
  • The more recent silk is lighter but that is to be expected because it is more dilute. (12 ml 3% SDS vs. 0.75 ml)