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ENZYME SUBSTRATES

Frequently Asked Questions on Enzyme Substrates

The intact substrate shows considerable fluorescence. Can I use it?

Enzymatic cleavage of fluorogenic carboxypeptidase substrates merely causes a shift of the excitation and fluorescence maxima to higher wavelengths e.g. AMC-substrates before cleavage λex ca 340 nm, λem ca 390 nm released 7-amino-4-methylcoumarin λex 360-380 nm, λem 440-460 nm

What is the difference between AMC and AFC substrates?

AFC substrates absorb and emit at higher wavelengths:
-released 7-amino-4-trifluoromethylcoumarin λexc 395-400 nm, λem 495-505 nm
-released 7-amino-4-methylcoumarin λexc 360-380 nm, λem 440-460 nm
Please see also our Technical Note “Chromophors/Fluorophors: Spectral Properties and Characteristics” which can be downloaded from our homepage.

What can I do to minimize background absorption of pNA substrates?

pNA substrates are hydrolyzed quite readily in aqueous buffers, hydrolysis is accelerated by increasing the pH. DRY organic solvents (e.g. DMSO, methanol) are best suited for preparing stock solutions, aliquotation is highly recommended. When diluting an aliquot with aqueous buffer, it should be kept cold and consumed during the working day.

What do you mean by FRET substrates?

Peptidic FRET (Fluorescence Resonance Energy Transfer) substrates contain a donor (a fluorophore) and an acceptor (quencher) which absorbs at the emission wavelength of the fluorophore.
The quencher absorbs the energy emitted by the fluorophore only as long as they are in close proximity, connected by the peptide chain. Enzymatic hydrolysis causes an increase of fluorescence irrespective of the cleavage position, provided that donor and acceptor are disconnected.
Bachem offers a large choice of FRET substrates for MMPs, ACE, β-secretase and other enzymes. For an overview of our products you can download our product monograph “FRET Substrates”.

Which FRET pairs do you offer?

Abz/various quenchers
N-Me-Abz/Dnp
EDANS/DABCYL
Mca/Dnp
Trp/Dnp

Do you offer a substrate for…

Entering the enzyme name in the search function could already return you a number of suitable substrates. Or click on the tab “Inhibitors & Substrates” and click on the name of the enzyme for which you require a substrate in the menu on the left-hand side.

 

In most cases, the enzymes are designated according to the BRENDA enzyme data base.

Do you offer an inhibitor for …

Click on the tab “Inhibitors & Substrates” and click on the name of the enzyme for which you require an inhibitor.

 

In most cases, the enzymes are designated according to the BRENDA enzyme data base. Or enter the enzyme name into the search field.

Can I search my dye labeled FRET substrates by wavelength?

Yes. Use our Dye Selector to search our dye labeled peptides including a broad selection of FRET substrates. You can select the absorption/excitation wavelength you are aiming for and our catalog products in this wavelength range will be displayed. Furthermore, you can specifically select specific dyes or filter the search results by research interests.