Firefly Sample Preparation  (note that uL = microlitres, ug = micrograms)


Cell Lysis Buffers + protease and phosphatase inhibitors

HNTG: 25mM Hepes (pH 7.5), 25mM NaCl, 0.1% triton, 10% glycerol [HNT(0.1%)G],
protease and phosphatase inhibitors as below.

RIPA: 25mM Hepes pH 7.5, 150mM NaCl, 1% NP 40, 0.25% Na-deoxycholate, 10% glycerol,
protease and phosphatase inhibitors as below.

Phosphatase Inhibitor Cocktail 1
Sigma-Aldrich (Cat# P2850),  $153.50/ 5mL bottle
1/100 is Working dilution

Protease Inhibitor
Calbiochem (Cat# 539134) or from VWR (Cat# 80053-852), $57.00
1/1000 is Working dilution


Cell Lysis Procedure for Adherent Cells
LNCaP lysate was prepared as follows from 10cm Petri dish at >90% confluence:

1.      Rinse cells twice with 5 mL ice cold HNG (25 mM Hepes, pH 7.5, 25 mM NaCl,
10% glycerol).
2.      Remove HNG completely by aspiration and lyse cells with 500 uL HNTG (or RIPA as you prefer).
3.      Incubate 10 min on ice with occasional swirl to cover cells.
4.      Scrape cells with rubber spatula or cell scraper into HNTG (or RIPA).
5.      Transfer cell suspension into microfuge tubes.
6.      Extract at 4°C for 1 hr on a nutator.
7.      Clear lysates by centrifuging 2x at 14000 x g /10 min / 4°C. SAVE
supernatants, transfer to fresh tube.
8.      Aliquot lysates into 10 uL volumes and store -80°C


Cell Lysis Procedure for Suspension Cells
Lyse cell pellets (3 million cells/pellet):

1.      Resuspend in 200 uL ice cold HNG (25mM Hepes (pH 7.5), 25mM NaCl, 10% glycerol)
2.      Spin down 3 min/ 1000 x g, discard supernatant and retain cell pellet.
3.      On ice, add 200 uL RIPA or HNTG lysis buffer.
4.      Incub on ice 10 min.
5.      Extract 4°C /1 hr rotating or rocking (Nutators are great).
6.      Clear lysates by spin 2x at 14000 x g / 4°C /10 min. SAVE supernatants,
transfer to fresh tube.
7.      Supernatants aliquotted 20 uL each and store -80°C


BCA (bicinchoninic acid) assay to quantitate total protein in lysates
Use Pierce BCA protein assay kit catalog #23225

a.      Make stds (one set for each type of lysis buffer used):

        tube ID uL lysis   uL      BSA     ug/mL
                buffer     BSA     source   BSA
               (=diluent)                  [final]
        A       0          50      stock        2000
        B       25         75      stock        1500
        C       50         50      stock        1000
        D       50         50      tube B       750
        E       50         50      tube C       500
        F       50         50      tube E       250
        G       50         50      tube F       125
        H       40         10      tube G       25
        I       50          0      ---          0

b.      Example of Unknowns:

        sample #   time pt (min)   lysed in
        1          0               HNTG
        2          5               HNTG
        3          15              HNTG
        4          30              HNTG
        5          60              HNTG
        6          0               RIPA
        7          5               RIPA
        8          15              RIPA
        9          30              RIPA
        10         60              RIPA

c.      Make working reagent (e.g., 40 wells = 10 unknowns in duplicate + 20 stds)
d.      Calc vol of Reagent A: 40 * 200 uL = 8000 uL A
e.      Calc vol of Reagent B: 8000 uL A / 50 = 160 uL B
f.      Working reagent = Reagent A+B = 8000 uL + 160 uL = 8160 uL. Soln turns green.
g.      Pipet 10 uL std or unknown into 96 well plate (black w/clear bottom Costar 3603)
h.      Pipet 200 uL working reagent into each well.
i.      Cover plate and shake 30 sec.
j.      Incub in cell culture incubator 30 min 37°C
k.      Remove from incub and allow to equilibrate to RT.
l.      Read plate in spec, endpoint read (optimal at 562 nm)