need help with calcium phosphate gel - (Apr/11/2006 )

Hello

I am extracting an enzyme from Gluconobacter using a paper published about 7 years ago. I've been following the methods for purification and am still getting nothing. Of course, the paper doesn't have much on specifics with spin times and speeds so i've been trying to get those dialed in with research on the net.

There is one part of the paper i can not find much on:

"10% CaCl2 solution was added to the clear supernatant containing the (enzyme) up to 0.5% final concentration. The calcium phosphate gel with the adsorbed (enzyme) was collected by centrifugation at 6000g..."

I have never used a calcium phosphate gel before and am a bit confused. I have been adding the CaCl2 into the tube and goin straight to the centrifuge, spinning down, and keeping the pellet. Does this sound right?

Also, the protocol has 2 ammonium sulphate steps. The first discards the precipitate, the second keeps the precip. After the second, my precipitate is still in solution after 15minutes at 15000g spin. Any suggestions?

Cheers,
dez

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yes. caphos gel adsorbs proteins (similar to hydroxylapatite, which is calcium phosphate hydroxide). you can elute the protein from the gel by adjusting pH or however they do it in the paper.

for the ammonium sulfate fractionation, ensure that all of the as is in solution then allow to sit, on ice, for at least an hour (because you are having difficulty getting it to pellet). also, make sure that you are using the correct amount of as in the first cut.

but, most important, since this is new to you, don't discard anything. you can resuspend, dialyze and reconstitute from any step in which you may have lost your protein.

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Thanks for the response. with that caphos gel, why is the supernatant looking so much like a gel as well? the pellet is a white 'cake'. for the 'as' i found a table for the amount needed to reach a percent. the first is 35% and the second is 65%

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when caphos first forms it is a white precipitate. i assume that as it ages (days, weeks, months?) it will become more "gel-like". i've used caphos gel in the procedure to purify urease. there, you add the gel to the protein solution but you are adsorbing contaminating proteins rather than the protein of interest. i wouldn't worry about the pellet's appearance, unless the procedure says something different.

as for the ammonium sulfate, does the table give you differential amounts. the first should be 0-35% and the second should be 35-65%. if the table doesn't give it to you that way then you have to measure the volume of the supernate after the 35% cut, determine the amount of as to make that volume 65%, then subtract the amount that you added to make 35% from the value for 65%.

if you added another 65%, that would make the solution 100% saturated ammonium sulfate and that would make the solution very dense. the pelleting would have to be done at a higher rcf and for a longer period of time.

hope this helps some

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Yeah, the table is like an old multiplication table, where you take initial concentration and match up with desired and it gives an amount to add. Thank you for the information and help. It's very much appreciated.

Cheers!
dez

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happy to be able to be of assistance.

mike

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