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micro micro
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10 years ago
If you treat cells with lysozyme/an enzyme to lyse the cell and obtain protein cell lysate to perform electrophoresis, how does one get rid of the lysozyme so it doesn't appear on the gel? Also, if boiling is used to denature proteases, how come the other proteins are not denatured?
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#1
Staff Member
Educator
10 years ago
Quote
If you treat cells with lysozyme/an enzyme to lyse the cell and obtain protein cell lysate to perform electrophoresis, how does one get rid of the lysozyme so it doesn't appear on the gel?

The lysozyme would have a known size.

Quote
if boiling is used to denature proteases, how come the other proteins are not denatured?

Once heat is removed, the proteins will renature.
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micro Author
wrote...
#2
10 years ago
Proteases are proteins too so wouldn't they be renatured as well rendering boiling useless?
wrote...
#3
Educator
10 years ago
Hi Micro, I don't remember ever having to remove the enzyme before running the same. The enzyme will have a distinct size once it runs through the gel.

Why are you boiling the same to denature it? Isn't there a better technique? Furthermore, why are you worried about whether it denatures? That won't effect the size of the sample. Denature means it loses its shape only, not mass.
micro Author
wrote...
#4
10 years ago
Boiling is carried out to inhibit the action of proteases / other degrading enzymes. Obviously adding other agents  to prevent protein degradation by enzyme action can be better but more expensive Wink Face
wrote...
#5
Staff Member
Educator
10 years ago
Quote from: micro (10 years ago)
Boiling is carried out to inhibit the action of proteases / other degrading enzymes. Obviously adding other agents  to prevent protein degradation by enzyme action can be better but more expensive Wink Face

We used to put the content in a heating bath at 65 degrees usually. It's the least expensive method.
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