Hi Liliana upon receipt the enzyme should be dispensed into small aliquots (2 μl) and stored at -80°C for avoiding repeated freezing and thawing. The stability of diluted enzyme can't be established excactly, therefore I use diluted enzyme immediately or within 1 week, storing it at -80°C. Alternatively since fpg is less stable than endo III someone suggests to prepare a stock Fpg solution (100x) using a buffer containing 10% glycerol and store 10 μl aliquots at -80°C. Then for use, dilute this aliquot to the final concentration with buffer wo glycerol but don't refreeze it.

The enzyme's final concentration to be used must be optimized for your particular cell line and type of exposure to maximize the difference in comet size between cells treated with enzyme and those exposed to reaction buffer only. So I suggest you to perfom an enzyme titration considering the undiluited enzyme and different enzyme diluition in reaction buffer (1:2, 1:5 ..)

Remember to include a buffer-only control, and if you treat your cells with some drug or radiation you should include a samples of untreated cells incubated in presence of enzyme, both as control for endogenous levels of damage within cells and damage that may occur during sample preparation.

Enzyme reaction buffer for endonuclease III and FPG

40 mM HEPES

0.1 M KCl

0.5 mM EDTA

0.2 mg/ml BSA

pH 8.0 with KOH

(can be made as 10 x stock, adjusted to pH 8.0 and frozen at -20°C)

I usually utilize a FLARE Assay Kit by trevigen

B

Thank Barbara for your help!

The enzymes that I've buyed are at a concentration of 10000 U/mL (EndoIII) and 8000 U/mL (Fpg) and I'm using gelbond film, it means that the films are incubated in 50mL of enzyme + buffer and I dont know which is the best way to aliquot them to store and which could be the range of the final concentration in these 50mL. Do you have any idea? I do not want to waste even one microliterof the enzymes. I'm using isolated and frozen lymphocites.

I greatly appreciate your collaboration and your time.

Hi Liliana, here I am..

As I wrote to you in the last message, I usually utilize Fpg FLARE™ Assay Kit to perform comet assay with enzyme.

The Fpg enzyme is among materials supplied and its concentration is 500 Units/150 μl (approximately 3.3 U/ μl). I also use Trevigen's 3 well FLARE™Slides and each sample area on the FLARE Slide requires 75 μl of working Fpg Enzyme, so according to Trevigen instructions I prepare the Working Fpg Enzyme Solutions as follows:

For 1 sample area:

- Fpg FLARE™ Reaction Buffer 75 μl

- Fpg Enzyme (e.g. straight or 1:2, 1:5 dilution) 1 μl

Then I immediately add 75 μl of working Fpg Enzyme Solution to each sample area.

The first time a possible scheme could be (for my protocol) :

e.g . For sample area 1 of FLARE™ Slide, apply buffer only; sample areas 2, undiluted Fpg; and sample area 3, 1:2 dilution of Fpg.

Obviously the enzyme’s final working concentration will change depending on your cell type, treatment condition and so on.. thereby I suggest you to make an enzyme’s titration for choosing the right concentration.

I’m inexperienced in come assay protocol with gelbond film…but even if I work with small volumes of solution you can calculate my final concentration of enzyme and after you will be able to set up your protocol: e.g the enzyme aliquots, the working enzyme concentration…

Attached a paper that describes fpg comet assay with gelbond film..you could contact the author for more information

B