Buffers, chemicals
Contact: Dr. Christos Chinopoulos MD, PhD chinopoulos.christos@eok.sote.hu


  M.W. final concentration (mM) 1000 ml Origin
KCl 74.55 8 596.4  mg 104938
MERCKMillipore

do NOT use other type of KCl
K-gluconate 234.25 110 25.767 gr G4500 SIGMA
  NaCl 58.44 10 584.4  mg S9888 SIGMA
Hepes (free acid) 238.3 10 2.383 gr 54457 SIGMA
K2HPO4 174.18 10 1.7418 gr 17835 SIGMA
EGTA (K+ salt) 380.35 0.015 5.7053 mg 03779 SIGMA
BSA (FA-free) - 0.5 mg/ml 500 mg A6003 SIGMA
Mannitol 182.2 10 1.822 gr M9546 SIGMA
  pH= 7.25 (using KOH-ultrapure or HCl-ultrapure) - - KOH:  P5958 SIGMA (make a
5N stock)
HCl: 339253 SIGMA (make a
5M stock)
'ANT buffer'
Just before the experiment, add MgCl2. For isolated mitochondria, use 1 mM final concentration; for permeabilized cells, 1.5 mM (unless you calibrate the magnesium green signal, or determine Kds of ADP or ATP for Mg2+, see under 'Kds determination of ADP, ATP to Mg2+').

MgCl2: M1028 SIGMA (1 M stock solution).

Do NOT keep buffer containing MgCl2 for more than one day!

final concentration Stock Solvent Origin
Magnesium Green, pentapotassium salt 1.1 mM 1.1 mM Water (add 993 ml of water in 1 mg vial); make at least 10 aliquots;
store in -20 oC
M3733 Thermo Fisher
Digitonin custom ≥2.5 mM DMSO. Aliquot and
keep in -20 oC
custom; best is from
Wako Chemicals:
Cat-no: 043-21371
Succinate 5 mM 0.5 M Water, pH 7.0-7.2 (KOH) 14080 SIGMA
Glutamate 5 mM 1 M Water, pH 7.0-7.2 (KOH) 49449 SIGMA
a-Ketoglutarate 5 mM 1 M Water, pH 7.0-7.2 (KOH) 75890 SIGMA
Malate 5 mM 0.5 M Water, pH 7.0-7.2 (KOH) 02288 SIGMA
do NOT use other
type of malate
Pyruvate 5 mM 1 M Water, pH 7.0-7.2 (KOH) P2256 SIGMA
EDTA custom; no less
than 10
mM
0.5 M - 03690 SIGMA
BeSO4*4H2O see below 0.2 M Water; do not adjust pH 14270 SIGMA
NaF see below 0.5 M Water; do not adjust pH S7920 SIGMA
Na3VO4 preparation needed
see below
25 mM preparation needed
see below
450243 SIGMA
Safranin O see below 1 mM Water; store at RT; do
not shake, allow precipitate to form;
always take from the top;
do not adjust pH
S2255 SIGMA
SF6847 Custom; maximum
concentration is 250 nM for complete depolarization of isolated mitochondria
1 mM Ethanol BML-EI215
ENZO Life Sciences
Ap5A 50 mM 0.1 M Water; do not adjust pH D4022 SIGMA
carboxyatractyloside 1 mM 2 mM Water; do not adjust pH 216201
MERCK Millipore
valinomycin 5 nM 1 mM Ethanol V0627 SIGMA
ADP (K+ salt)* custom 0.2 M Water. pH between
6.2 and 6.9. Freeze to
-20 oC in aliquots. Aliquots can be reused
up to 3 times.
117105
MERCK Millipore

do NOT use other
type of ADP
ATP (diK+ salt)* custom 0.2 M Water. pH between
6.2 and 6.9. Freeze to
-20 oC in aliquots. Aliquots can be reused
up to 3 times
A7699 SIGMA
do NOT use other
type of ATP
EGTA solution custom 0.2 M Water; pH to 7.0 03779 SIGMA
oligomycin 10 mg/ml 10 mg/ml Ethanol O4876 SIGMA
Other chemicals required, depending on the experimental scheme:
Preparation of sodium orthovanadate (Na3VO4) and beryllium trifluoride (BeF3-): A 25 mM Na3VO4 solution is prepared in distilled water (>17 megaOhm resistance). The pH of the solution is adjusted to 8.7 with HCl, upon which it turns yellow. This solution is boiled until it turns colorless and cooled to room temperature. The pH is reassessed, and readjusted to pH 8.7 with HCl, upon which the solution turns yellow again. This cycle of boiling until colorless and readjusting the pH is repeated until the solution remains colorless at pH 8.7.  Finally, the solution is brought up to the initial volume with distilled water and stored in aliquots at -20 °C. This treatment removes all decavanadate ions present in the Na3VO4 solution, which induces mitochondrial membrane depolarization and inhibition of oxygen consumption (Aureliano and Crans 2009). Orthovanadate inhibits the oxidation of only disrupted mammalian mitochondria (Byczkowski et al. 1979).

Likewise, fluoroberyllium nucleoside diphosphate complexes inhibit only the exposed F1F0-ATPase (Issartel et al. 1991). BeSO4 and NaF are prepared as aqueous stock solutions of 0.2 M and 0.5 M, respectively, and kept at +4 °C for several years. BeF3- is formed immediately in solution upon mixing of BeSO4 and NaF, provided that NaF is in excess in the working solution. BeF3- is formed immediately in solution upon mixing of 2 microliters of 0.2 M in a 2 ml cuvette volume containing the buffer of your choice (i.e. 200 micromolar BeSO4 final concentration) and 20 microliters of 0.5 M NaF, (i.e. 5 millimolar NaF final concentration). As such, final concentration of BeF3- will be 200 micromolar. 

Vanadate, beryllium and fluoride salts are highly toxic to tissues and to the environment, and thus require proper handling and disposal. The combination of orthovanadate and BeF3- will inhibit kinases, mutases, phosphatases, and ATPases (Ray et al. 1990; Climent et al. 1981). However, some kinases, such as pyruvate kinase, will remain uninhibited (Lord and Reed 1990). In this respect, upon permeabilization of the cells one has to totally separate pyruvate kinase from its substrate, phosphoenol pyruvate, i.e. there must be no glucose present in the medium prior to permeabilization, and a few minutes lag time must be allowed prior to ADP-ATP exchange rate measurements in order for the remaining reactions by kinases to ‘die-out’.

*Concentration of ADP and ATP stock solutions is corrected by measuring absorbance at 260 nm using an extinction coefficient eM= 15,400 M-1*cm-1.