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What should be the goal amount of protein to get from purification?


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#1 Sharky

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Posted 19 April 2010 - 06:07 AM

Just a simple question. I am trying to purify an enzyme(wild type) to do biochemical characterization. How much mg of protein should I aim to have to get data for enzyme assay kinetics/thermostability data without running out of enzyme? Because I see the His trap columns only come for 20ml ? But people tell me to make culture in 2 litres?! I am confused.

#2 ProteinWork

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Posted 19 April 2010 - 06:35 AM

Just a simple question. I am trying to purify an enzyme(wild type) to do biochemical characterization. How much mg of protein should I aim to have to get data for enzyme assay kinetics/thermostability data without running out of enzyme? Because I see the His trap columns only come for 20ml ? But people tell me to make culture in 2 litres?! I am confused.

20ml is the bed volume of the column. You can pass more than that volume through the column since it's an affinity column and theoretically only your protein with his tag will bind to the column. So before the column is saturated with his-tag protein, you can do multiple loads of your sample.

And you do not directly load the 2-litre culture onto the column, either. You have to spin down the cells from the 2-liter culture and resuspend the cell pellet in a much smaller volume of lysis buffer, typically 30-50ml, and then go through a series of lysis steps till you get the lysate. Only then you shall load the lysate onto the column.

I'm sure whoever is guiding you should have a much detailed protocol than I've written above. Just read the protocol and your many questions will be answered.

#3 Sharky

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Posted 19 April 2010 - 07:32 AM

Oh ofcourse, I got severely confused. I get it now, haha. Cheers man!

Just a simple question. I am trying to purify an enzyme(wild type) to do biochemical characterization. How much mg of protein should I aim to have to get data for enzyme assay kinetics/thermostability data without running out of enzyme? Because I see the His trap columns only come for 20ml ? But people tell me to make culture in 2 litres?! I am confused.

20ml is the bed volume of the column. You can pass more than that volume through the column since it's an affinity column and theoretically only your protein with his tag will bind to the column. So before the column is saturated with his-tag protein, you can do multiple loads of your sample.

And you do not directly load the 2-litre culture onto the column, either. You have to spin down the cells from the 2-liter culture and resuspend the cell pellet in a much smaller volume of lysis buffer, typically 30-50ml, and then go through a series of lysis steps till you get the lysate. Only then you shall load the lysate onto the column.

I'm sure whoever is guiding you should have a much detailed protocol than I've written above. Just read the protocol and your many questions will be answered.



#4 Inmost sun

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Posted 19 April 2010 - 10:39 AM

Just a simple question. I am trying to purify an enzyme(wild type) to do biochemical characterization. How much mg of protein should I aim to have to get data for enzyme assay kinetics/thermostability data without running out of enzyme? Because I see the His trap columns only come for 20ml ? But people tell me to make culture in 2 litres?! I am confused.


I do not know if you will really get purified protein in mg range ( I once was happy to get one ng of a protein kinase but it was really pure); down scale your assay protocols to save enzyme




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