Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/29 July 2015"

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=Honeybee Inclusion Body Purification=
+
='''Honeybee Inclusion Body Purification'''=
 
#Pre weigh two 50 ml falcon tubes
 
#Pre weigh two 50 ml falcon tubes
 
#Split the culture into two 50 ml falcon tubes.
 
#Split the culture into two 50 ml falcon tubes.
Line 292: Line 292:
 
#Decant supernatant, and make sure to get rid of as much liquid as possible.
 
#Decant supernatant, and make sure to get rid of as much liquid as possible.
 
#Record the weight of the pellets.  
 
#Record the weight of the pellets.  
 +
#*In total, the two pellets weighed 0.67 grams, which is lower yield than last time.
 
#Transfer pellet to one falcon tube.
 
#Transfer pellet to one falcon tube.
 
#Resuspend in 5 ml/g of pellet Bug Buster (1x) by pipetting and gently vortexing.
 
#Resuspend in 5 ml/g of pellet Bug Buster (1x) by pipetting and gently vortexing.
 
#Put on shaker or rotating mixer for 15 min at RT.
 
#Put on shaker or rotating mixer for 15 min at RT.
−
#*Take first fraction, F1 of this full cell lysate.
+
#*Take first fraction, (F1) of this full cell lysate.
 
#Centrifuge 16000 g 20 min at 4 degrees C
 
#Centrifuge 16000 g 20 min at 4 degrees C
−
#*Take second fraction of this supernatant which should contain soluble proteins, while our desired protein should be in the pellet.
+
#*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 as above.
 
#Resuspend in the same volume of 1X Bugbuster as above.
 
#*Make sure the pellet is completely resuspended!
 
#*Make sure the pellet is completely resuspended!
 
#*Take Fraction at this point (F2) Cell lysate #2
 
#*Take Fraction at this point (F2) Cell lysate #2
−
#Add DNAse (1 ul) and let rotate 20 min.
+
#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
 
#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.
 
#*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)#*
 
#Add 6 volumes of 1:10 diluted bugbuster (.1X)#*
 +
#*6x3.4= 20.4 ml
 
#*Can split up into two falcon tubes if necessary.
 
#*Can split up into two falcon tubes if necessary.
 
#Centrifuge 16000g 15 min. 4 degrees C to collect inclusion bodies.
 
#Centrifuge 16000g 15 min. 4 degrees C to collect inclusion bodies.
−
#Collect supernatant as fraction S2. Inclusion body should be the pellet.
+
#Collect supernatant as fraction (S2). Inclusion body should be the pellet.
 
#Resuspend pellet in 1/2 volume of original 0.1X bug buster solution
 
#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.
 
#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.
+
#*Take supernatant fractions for each wash step. (s3)
−
#Repeat step 15-16 two more times.
+
#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.
 
#Resuspend inclusion body pellet in 3% SDS solution and incubate at 60 C in the water bath for 2 hours.
 +
#*Used 750 ul SDS. (Final)
 
#Store at 4C for further processing and analysis.
 
#Store at 4C for further processing and analysis.

Latest revision as of 21:01, 24 August 2015

Honeybee Inclusion Body Purification

  1. Pre weigh two 50 ml falcon tubes
  2. Split the culture into two 50 ml falcon tubes.
  3. Mass balance and spin down cells at 5300 rpm 4C for 15 minutes.
  4. Decant supernatant, and make sure to get rid of as much liquid as possible.
  5. Record the weight of the pellets.
    • In total, the two pellets weighed 0.67 grams, which is lower yield than last time.
  6. Transfer pellet to one falcon tube.
  7. Resuspend in 5 ml/g of pellet Bug Buster (1x) by pipetting and gently vortexing.
  8. Put on shaker or rotating mixer for 15 min at RT.
    • Take first fraction, (F1) of this full cell lysate.
  9. 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.
  10. Resuspend in the same volume of 1X Bugbuster as above.
    • Make sure the pellet is completely resuspended!
    • Take Fraction at this point (F2) Cell lysate #2
  11. Add DNAse (2 ul) and let rotate 20 min. (This may have been too much DNAse but it should be ok.)
  12. 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.
  13. Take another fraction (F3) here, to see if there is a difference after adding the DNAse and the lysozyme.
  14. Add 6 volumes of 1:10 diluted bugbuster (.1X)#*
    • 6x3.4= 20.4 ml
    • Can split up into two falcon tubes if necessary.
  15. Centrifuge 16000g 15 min. 4 degrees C to collect inclusion bodies.
  16. Collect supernatant as fraction (S2). Inclusion body should be the pellet.
  17. Resuspend pellet in 1/2 volume of original 0.1X bug buster solution
    • 10.2 ml
  18. 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)
  19. 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.
  20. Resuspend inclusion body pellet in 3% SDS solution and incubate at 60 C in the water bath for 2 hours.
    • Used 750 ul SDS. (Final)
  21. Store at 4C for further processing and analysis.