Furin - molbio methods

Furin expression

Furin sold by NEB: Furin

(Fugere Day 2001 pmid: 11723118) stable schneider cells expressing SPC1delta (lacking the transmembrane domain) = Furin

(Komiyama Fuller 2000 pmid: 11106495) soluble Furin as baculo virus (asked for sending 19-2-2020) - Q-sepharose purification

Than lab - asked for transient expression system.

Furin assay

(Hatsuzawa Nakayama 1992 pmid: 1644796) Furin assay: Boc-Arg-Val-Arg-Arg-AMC 4018735 | Bachem https://shop.bachem.com/4018735.html - Stock solutions of Boc-RVRR-AMC are best prepared in DMSO. -

(t-butoxycarbonyl-Arg-Val-Arg-Arg-4-methylcoumarin-7-amide) Lösen - 843 gr/mol; 25 mg sind 29,7 micromol; Stocklösung 100mM in DMSO - lösen in 297ul (check again please)

100mM entspricht 100nmol/ul - daruf achten, dass die DMSO Konzentration im Assay nicht zu groß wird (<1%?)

Samples were incubated with Boc-Arg-Val-Arg-Arg- MCA (20 nmol) in a final volume of 250ul of 100 mM MES/NaOH, pH 7.0, containing 1 mM CaCl2 at 37°C for 1-4 h. The reaction was stopped by the addition of 3 ml of 5 mM EDTA, and the amount of MCA released from the substrate was measured in a fluorescence spectrophotometer with excitation at 380 nm and emission at 460 nm. (535 geht gerade noch) (geht auch mit geringeren Mengen und Volumen - Gerät von Sprangers oder Grohmann)

our assay: 20 ul / 5nmol BOC / Furin: verschiedene Konzentration 1u / 0.1 / 0.01u / Tris Puffer + wie NEB Puffer

Furin from NEB

Isolated from Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus carrying truncated human furin (kindly provided by R. Fuller)

NEB assay conditions

One unit is defined as the amount of Furin that will release 1 pmol of AMC from the fluorogenic peptide BOC-RVRR-AMC (Bachem #I-1645) in one minute (1 pmol of AMC/min) at 30°C.

Unit Assay Conditions: 100 mM HEPES (pH 7.5 @ 25°C), 0.5% Triton X-100, 1 mM CaCl2, 1 mM 2-mercaptoethanol, 100 μM B0C-RVRR- AMC and enzyme in a 100 μl volume at 30°C.