Amylase and Pepsin Preparaton

defencegrid

wrote...

Posts: 8

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7 years ago

How would I prepare 1% solutions of Amylase and Pepsin?

With the Amylase is it just a matter of dissolving 1g of powder in 100ml of DI water?, or do you need to dissolve it in pH7 buffer?

With the Pepsin, do I need to add 1g of Pepsin to let's say 90ml of DI water, then add HCL drop wise to bring the pH down to about two, and then make it up to 100ml with water?

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bio_man

wrote...

Educator

7 years ago

Use distilled water. You're on the right track.

As for pepsin, take a read here: http://science.jburroughs.org/resources/MBLabs/16_Protein_Digest_Teach2.pdf

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defencegrid Author

wrote...

7 years ago

Quote from: bio_man (7 years ago)

Use distilled water. You're on the right track.

As for pepsin, take a read here: http://science.jburroughs.org/resources/MBLabs/16_Protein_Digest_Teach2.pdf

So from the above document, it says to just add the Pepsin powder to DI water, so I don't need to acidify the solution?

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bio_man

wrote...

Educator

7 years ago

Nope

Could you explain the logic behind your thinking?

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defencegrid Author

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7 years ago Edited: 7 years ago, defencegrid

Well pepsin works best at a pH of abut 2, so I'm thinking you need to add water to the pepsin powder and then add HCL to bring the pH down.
.

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bio_man

wrote...

Educator

7 years ago

I disagree. I think you should leave it the way it is, without adding HCl

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defencegrid Author

wrote...

7 years ago

Quote from: bio_man (7 years ago)

I disagree. I think you should leave it the way it is, without adding HCl

So you can just prepare it at pH7?

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bio_man

wrote...

Educator

7 years ago

Yes. It will activate when the solution is at the most appropriate for the enzyme

Now I'm second guessing myself! Undecided

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defencegrid Author

wrote...

7 years ago

This document https://assist.asta.edu.au/print/2816 says that Pepsin requires a pH of 1.5-2.0 to be active.

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bio_man

wrote...

Educator

7 years ago

I understand but if you're mixing it into a medium that's already acidic, you wouldn't need to use HCl. I think the main reason I'm second guessing myself is because I don't know what the purpose of the experiment is, and I'm not even sure what you're trying to accomplish. I'm assuming you want to observe how pepsin digests protein, but that's the scoop that I know

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defencegrid Author

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#10

7 years ago

Quote from: bio_man (7 years ago)

Yes observe how pepsin digests protein. I don't know what you mean by the medium that I'm mixing the pepsin into.

The medium is just water.

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bio_man

wrote...

#11

Educator

7 years ago

Yes, so in that case if you're testing the effect of pepsin when mixed with meat, you need to use HCl instead of distilled water. You initial post was correct, in terms with f methodology. What's the percentage of your stock HCl solution?

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defencegrid Author

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#12

7 years ago

Well that's also what I'm not sure about. I was thinking of using 1M HCL drop wise until the pH drops to about pH2.

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bio_man

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#13

Educator

7 years ago

I'm not sure how you'd find the concentration of an unlabeled solution of HCl, but every stock solution is usually labeled. Molarity is a measure of moles of solute over the volume of the solution. You could try that method, absolutely

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