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Preparation of Purified Inclusion Bodies


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#1 mmmm

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Posted 14 October 2010 - 11:13 PM

hi all,
I just have one problem in this above procedure.i m not getting pellet on centrifugation properly..the supernatant is not clear and remains like a jelly solution which i cant seperate from the pellet properly.. what might be the problem..is it due to old sucrose.. please suggest....






Thaw cell pellets on ice and resuspend each pellet in 4 mL buffer per gram cell pellet
(buffer made freshly b/c contains dTT):
500 mL IB extraction buffer:
20 mM Tris-HCl (157.6 g/mole)
2 mM EDTA (292.25g/mole)
3 mM dTT (154.2 g/mole)
pH 7.5
Lyse cells three times
Centrifuge lysate over 10 mL of 27% sucrose at 8,000 rpm for 45 min.
Resuspend the pellets in 2-3 mL IB extraction buffer
Lay 2-3 mL of resuspended pellet on 10 mL sucrose again and centrifuge
as before.
Resuspend the pellet in 500 uL of IB extraction buffer, Split into 2
pre-weighed microfuge tubes, and spin for 7 minutes at 13,000 rpm in a microcentrifuge.

Thanks in advance.

#2 protolder

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Posted 15 October 2010 - 01:44 AM

hi all,
I just have one problem in this above procedure.i m not getting pellet on centrifugation properly..the supernatant is not clear and remains like a jelly solution which i cant seperate from the pellet properly.. what might be the problem..is it due to old sucrose.. please suggest....






Thaw cell pellets on ice and resuspend each pellet in 4 mL buffer per gram cell pellet
(buffer made freshly b/c contains dTT):
500 mL IB extraction buffer:
20 mM Tris-HCl (157.6 g/mole)
2 mM EDTA (292.25g/mole)
3 mM dTT (154.2 g/mole)
pH 7.5
Lyse cells three times
Centrifuge lysate over 10 mL of 27% sucrose at 8,000 rpm for 45 min.
Resuspend the pellets in 2-3 mL IB extraction buffer
Lay 2-3 mL of resuspended pellet on 10 mL sucrose again and centrifuge
as before.
Resuspend the pellet in 500 uL of IB extraction buffer, Split into 2
pre-weighed microfuge tubes, and spin for 7 minutes at 13,000 rpm in a microcentrifuge.

Thanks in advance.

Hola one more time. Following your messages, I think you are trying reproduce any standard protocolof your lab. I think that the viscosity of the bacterial suspension is due to the high density of 1g/4ml, and the partial lysis with the DNA unbroken. So try to sonicate (Your inclusion bodies resist) to broke DNA or increase the volume of sucrose solution and speed. More things if in your protocol,the protein of interest remain soluble in 8M Urea, centrifugue analize pellet and continue with clean supernatant.More things never heat utea solution to dissolve, it coul form cyanide and more unwhised compounds. Good luck

#3 mmmm

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Posted 15 October 2010 - 03:56 AM


hi all,
I just have one problem in this above procedure.i m not getting pellet on centrifugation properly..the supernatant is not clear and remains like a jelly solution which i cant seperate from the pellet properly.. what might be the problem..is it due to old sucrose.. please suggest....






Thaw cell pellets on ice and resuspend each pellet in 4 mL buffer per gram cell pellet
(buffer made freshly b/c contains dTT):
500 mL IB extraction buffer:
20 mM Tris-HCl (157.6 g/mole)
2 mM EDTA (292.25g/mole)
3 mM dTT (154.2 g/mole)
pH 7.5
Lyse cells three times
Centrifuge lysate over 10 mL of 27% sucrose at 8,000 rpm for 45 min.
Resuspend the pellets in 2-3 mL IB extraction buffer
Lay 2-3 mL of resuspended pellet on 10 mL sucrose again and centrifuge
as before.
Resuspend the pellet in 500 uL of IB extraction buffer, Split into 2
pre-weighed microfuge tubes, and spin for 7 minutes at 13,000 rpm in a microcentrifuge.

Thanks in advance.

Hola one more time. Following your messages, I think you are trying reproduce any standard protocolof your lab. I think that the viscosity of the bacterial suspension is due to the high density of 1g/4ml, and the partial lysis with the DNA unbroken. So try to sonicate (Your inclusion bodies resist) to broke DNA or increase the volume of sucrose solution and speed. More things if in your protocol,the protein of interest remain soluble in 8M Urea, centrifugue analize pellet and continue with clean supernatant.More things never heat utea solution to dissolve, it coul form cyanide and more unwhised compounds. Good luck


Thank you very much for your continuous suggestions. ya as you said i am following a standard protocol. will try as you said by sonicating for a long time.




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