UV-VIS Spectroscopy question

[Lindsayyyy]

Hello everyone,

I'm quite new to lab work and I have the following question:

I'm doing immobilisation on enzymes and I want to quantify how much enzymes is bound to my material after immobilization. I'm using UV-VIS for this.

Before the experiment itself I want to a calibration with known enzyme concentrations.
After the immobilization I will measure the natant to see how much enzyme is immobilized.

I have a question concerning the blanks.

In my natant there are severel different compounds (I don't know the exact concentrations) but they don't absorb at the wavelength my protein does.
Can I compare my results when I use the same blank for my calibration measurement (for example some kind of buffer) as well as for my final measurement?

So basically I use a buffer for my calibration to zero my UV VIS and then I use the same zero for my final measurement, will this work?

Thanks for your help

 

[Borek]

If you are sure they don't interfere - it shouldn't be a problem. But they can interfere in other ways as well - for example somehow reacting with your enzyme and changing its extinction coefficient.

 

[Ygggdrasil]

To what type of material are you immobilizing your enzyme? Many plastics absorb UV, so they would interfere with your A280 measurements, and materials such as beads can scatter light and would also interfere with your absorbance measurement.

Another strategy would be to use a functional assay to determine the amount of protein you immobilize. For example, if your enzyme catalyzes a certain reaction, you can monitor the reaction performed by the immobilized protein to determine the amount of immobilized protein.

 

[epenguin]

I'm doing immobilisation on enzymes and I want to quantify how much enzymes is bound to my material after immobilization. I'm using UV-VIS for this.

Not realLy empugh information on what you are trying to do and how. The above sentence reads like you want to know how much protein is not immobilised. Look up on Google 'protein assay' or suchlike (burnet, Lowry, Bradford etc but proabably any lab in which this experiment is being done would have a kit for it) to find a procedure that hopefully the other substances will not interfere with - which you must however check experimentally.

At the end of the day you want to know if you have enough immobilised enzyme for whatever you want it for- so just measure its catalytic activity as has been said,

Then if you want, e.g. for thinking about how to improve, to understand more of what is happening, you might want to see how much is left in your supernatant. Measure that activity too then! Of course you will also do some experiment with enzyme plus linking reactants without the solid substrate to see how much these reactants inactivate the enzyme.