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Problem clarifying lysate from Hi5 insect cells


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#1 Kimberly Decker

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Posted 05 April 2010 - 06:18 PM

I am trying to express a protein from Hi5 cells using a baculovirus system. When I lyse these cells and centrifuge to clarify the lysate, I see a white film floating on top of the clarified lysate. In addition, the top half of the lysate is cloudy, with an opaque, slightly stringy precipitate. I cannot get this cloudy part of the lysate to pellet at the bottom of the tube and I can't easily separate the cloudy from clear portion of the lysate, without losing a great deal of my lysate (which contains my protein).

At this point, I've just tried to continue purification with the cloudy lysate but it really clogs up the column. I have tried filtering the lysate with a 0.22uM and 0.45uM filter. These filters work well to clear the lysate but clog up after about 1mL has passed through! I used a Millipore prefilter, which helps the situation and does not clog; however, it does not remove a significant amount of this precipate.

Has anyone experienced this problem or know what this viscous material might be? Carbohydrate or lipid, perhaps? I don't believe it's DNA as the lysate is DNAse-treated. Any help oir suggestions would be appreciated.

Thanks,
Kim Decker

#2 RMcKenney

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Posted 07 April 2010 - 10:26 AM

I am trying to express a protein from Hi5 cells using a baculovirus system. When I lyse these cells and centrifuge to clarify the lysate, I see a white film floating on top of the clarified lysate. In addition, the top half of the lysate is cloudy, with an opaque, slightly stringy precipitate. I cannot get this cloudy part of the lysate to pellet at the bottom of the tube and I can't easily separate the cloudy from clear portion of the lysate, without losing a great deal of my lysate (which contains my protein).

At this point, I've just tried to continue purification with the cloudy lysate but it really clogs up the column. I have tried filtering the lysate with a 0.22uM and 0.45uM filter. These filters work well to clear the lysate but clog up after about 1mL has passed through! I used a Millipore prefilter, which helps the situation and does not clog; however, it does not remove a significant amount of this precipate.

Has anyone experienced this problem or know what this viscous material might be? Carbohydrate or lipid, perhaps? I don't believe it's DNA as the lysate is DNAse-treated. Any help oir suggestions would be appreciated.

Thanks,
Kim Decker



Hello,

I too have experienced this with Hi5 cells, although the white stuff (I believe it is lipid) was not so significant in my case. I usually tried to avoid it when removing my supernatant although some of it always came with it. I would suggest increasing the volume of lysis buffer you are using to lyse the cells to see if you can cut down on the total volume taken up by the lipid layer on top. That would make it easier to avoid when removing your supernatant. If you are using affinity tags to purify your protein, an increase in initial volume shouldn't pose any problem since your protein of interest will be concentrated on the column after lysis.

Best luck,
Rick




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