BMP Luciferase Assay

(Zilberberg et al., BMC Cell Biol. 2007 Sep 19;8:41)
 
(A.) Reaction buffer for Luciferase Assay 
 
1. Buffer stock: it is usually most convenient to make 20 or so aliquots at once. After mixing, use 2 ml per individual aliquot of buffer stock. 
 
Reagent buffer:                        Stock Conc                        Final [ ]                      for 40 ml
Tricine (MW= 179 g/mole)            1 M (ph 7.8)                       15mM                              3 ml
MgSO4(MW=120 g/mole)             750 mM (0.9 g/10 ml)         7.5 mM                             2 ml
DTT (MW 154.25 g/mole)             200 mM (1.54 g/50ml)          5 mM                              5 ml
ATP (MW 551 g/mole)                 75 mM (0.413 g/10ml)         1.5 mM                             4 ml
 
Adjust to 40 ml with H2O. Adjust to ph 7.8
 
Aliquot in 20*2 ml 
 
2. Luciferin stock (BD Pharmingen # 51-556879, MW 302.3 g/mole): 
 
Make stock at 15 mM. Make aliquots of 500 ul. The final luciferin concentration will be 0.75 mM.
 
3. To make 10 ml of reaction buffer: Mix  2 ml buffer mix + 7.5 ml H2O  + 500 μl luciferin
 
(B.) The BMP assay was performed as follows: 
  1. C2C12BRA and HepG2BRA cells were plated in 96-well tissue culture dishes at 4 Χ 103 and 5 Χ 103 cells/well, respectively (in general depending of the experiment the right number of cells will have to be determine). The cells were allowed to attach overnight in DMEM 10 % serum (the time of attachment can also be modified). 
  2. Wash 1X with PBS or serum-free medium. Add samples. Incubation time generally should be 12-18 h. Incubation can be in regular or serum-free medium.
  3. After Incubation add 40 μl of 1X lysis buffer (BD Pharmingen Luciferase 3X Cell lysis buffer #51-556871) to each well. Lyse cells for 20 min at RT on shaker. (at this point you can cover samples with Parafilm and store frozen at –20 C until ready to assay).
  4. Get 96 well luminometer assay plate. Transfer 30 μl of the cell lysate to the new plate. 
  5. Measure luciferase activity. The luminometer injects 100ul of the reaction buffer solution in each well.
Tricine Sigma T9784-25G
DTT DL-Dithiothreitol Sigma D0632-10G
ATP Sigma # A-7699 
 
Cell line Designation C2C12BRA
  • Culture in DMEM 10% FCS
  • Selection with 700 ?g/ml G418
  • Avoid the cells to reach confluence (confluency less than 24h should be OK) and to differentiate into myotubes
Cell line Designation HepG2BRA
  • Culture in DMEM 10% FCS
  • Selection with 700 ?g/ml G418