2 replies to this topic

[avitas]

Hi,

I have problems filtering my Hi5 insect cell lysates when trying to purify intracellular protein. Could someone please give me some advice??

After lysing the cell pellet with lysis buffer (~1L culture in 100mls of buffer) I pipette them up and down to disburse the cells and sonicate briefly to break cells. I then spin down the mixture which removes most cell debris and tried filtering them through 0.45um vaccum filters, which clog up very fast and I had to go through 5 filters. Is there any way to clarify the lysates or any product out there for clarification?

Thanks in advance for any advice!!

[Kimberly Decker]

avitas, on Jun 15 2010, 01:25 PM, said:

Hi,

I have problems filtering my Hi5 insect cell lysates when trying to purify intracellular protein. Could someone please give me some advice??

After lysing the cell pellet with lysis buffer (~1L culture in 100mls of buffer) I pipette them up and down to disburse the cells and sonicate briefly to break cells. I then spin down the mixture which removes most cell debris and tried filtering them through 0.45um vaccum filters, which clog up very fast and I had to go through 5 filters. Is there any way to clarify the lysates or any product out there for clarification?

Thanks in advance for any advice!!

I express a protein and use Hi5 cells. Here is my protocol for purification:

* I lyse the cells in a little more lysis buffer than I normally might, which seems to reduce the "slime" a little. After spinning down the cells, I use a syringe and needle to pull up the clarified lysate (supernatant) from the pellet. Then, I filter it using a Millipore pre-filter. These have a larger pore size and are considerably cheaper than 0.45uM filters. I usually need ~2 per 50 mls.
* My firsrt purification is an GE ion exchange column. I run this purification on an akta, and simply do multiple, simultaneous 10mL injections onto a 5mL column. When I elute, the lysate is remarkably clean. I followup with purification on a StrepTrap column (my protein is Strep2Tagged), and sometimes size exclusion.
* In truth, the key to addressing this problem is not to worry too much about it. I spent weeks trying to find ways to get rid of the debris, but once I incorporated the pre-filters and moved on with purification as normal, everything worked great!

Hope that helps.

-KD

[avitas]

Kimberly Decker, on Jun 16 2010, 06:53 PM, said:

avitas, on Jun 15 2010, 01:25 PM, said:

Hi,

I have problems filtering my Hi5 insect cell lysates when trying to purify intracellular protein. Could someone please give me some advice??

After lysing the cell pellet with lysis buffer (~1L culture in 100mls of buffer) I pipette them up and down to disburse the cells and sonicate briefly to break cells. I then spin down the mixture which removes most cell debris and tried filtering them through 0.45um vaccum filters, which clog up very fast and I had to go through 5 filters. Is there any way to clarify the lysates or any product out there for clarification?

Thanks in advance for any advice!!

I express a protein and use Hi5 cells. Here is my protocol for purification:

* I lyse the cells in a little more lysis buffer than I normally might, which seems to reduce the "slime" a little. After spinning down the cells, I use a syringe and needle to pull up the clarified lysate (supernatant) from the pellet. Then, I filter it using a Millipore pre-filter. These have a larger pore size and are considerably cheaper than 0.45uM filters. I usually need ~2 per 50 mls.
* My firsrt purification is an GE ion exchange column. I run this purification on an akta, and simply do multiple, simultaneous 10mL injections onto a 5mL column. When I elute, the lysate is remarkably clean. I followup with purification on a StrepTrap column (my protein is Strep2Tagged), and sometimes size exclusion.
* In truth, the key to addressing this problem is not to worry too much about it. I spent weeks trying to find ways to get rid of the debris, but once I incorporated the pre-filters and moved on with purification as normal, everything worked great!

Hope that helps.

-KD

Thank you very much KD!!