Science Buddies: "Ask an Expert"

I'm doing this project:
https://www.sciencebuddies.org/science- ... p011.shtml

I've actually already done it successfully using the stated methods. What I'd like to do though is perform a better extract using higher level filtration by using 200 micron, 100 micron and maybe 10 or even 1 micron filter bags. I figured I could maybe hang a bag a 200 micron bag with the solution in it and collect the filtrate underneath, and then transfer that filtrate to the 100 micron bag, etc. The effectiveness of the experiment thereafter would give me a better understanding of the enzymes ability to diffuse across barriers.

The question I have though is related to step 3 of the catalase extraction where it says "From this point on, the enzyme preparation must be carried out in an ice bath." What is the purpose of the ice bath? Will extraction not work without it/is it absolutely necessary? I was able to do the extraction using low-level filtration in an ice bath, but I figure higher level filtration would take much more time, probably hours, and the ice bath portion might be more difficult to accomplish due to melting, among other concerns.

Hi,

The reason for keeping catalase solutions cold during isolation was stated in the write up:

“Because enzymes are proteins, they are somewhat fragile. They can be denatured by heat, and can easily be broken down by proteases when cells are homogenized. To preserve activity of proteins in solution, it is important to keep the solutions on ice until you are ready to use them. Denaturing conditions, such as boiling, can also be used as evidence to show that an enzyme-based reaction is protein-dependent.”

You will have better results if you follow it. Possibly the following article will be of use:

http://repositorium.sdum.uminho.pt/bits ... 3/1/44.pdf

Hope this helps!

Barrett Tomlinson