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Sticky Insect cell lysate blocked Ni_NTA column


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#1 Christ

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Posted 12 March 2009 - 01:17 PM

Hi folks,

We are working to purify a protein using baculovirus system. We broke Sf9 cells (2L) using sonication with 100 ml buffer. Then loading the 100 ml supernatant is hack of hell, it was very sticky and blocked the Ni-NTA column. What is a possible reason, and how to solve the problem? Thanks.

Christ

#2 T C

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Posted 12 March 2009 - 07:30 PM

Hey

Precipitating DNA by using 0.1 % poly ethylene imine helps in decreasing viscosity and increases binding.....also dilution may just help.

Best
TC

Hi folks,

We are working to purify a protein using baculovirus system. We broke Sf9 cells (2L) using sonication with 100 ml buffer. Then loading the 100 ml supernatant is hack of hell, it was very sticky and blocked the Ni-NTA column. What is a possible reason, and how to solve the problem? Thanks.

Christ



#3 klinmed

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Posted 13 March 2009 - 03:20 AM

Hi folks,

We are working to purify a protein using baculovirus system. We broke Sf9 cells (2L) using sonication with 100 ml buffer. Then loading the 100 ml supernatant is hack of hell, it was very sticky and blocked the Ni-NTA column. What is a possible reason, and how to solve the problem? Thanks.

Christ


A simple solution which works is to "batch bind" your protein to the resin. Incubate the lysate with the Ni-NTA resin in a tube(s) for 1-2 hours at 4oC with mixing on a rotator (not a magnetic bar!). Wash by gentle centrifugation/resuspension x2 , then pour column and wash/elute as normal. This usually works fine with viscous lysates.

In the past have also used nucleases (Benzonase) to "thin" the lysate. But batch-binding is now standard in my lab.

Edited by klinmed, 13 March 2009 - 03:21 AM.





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