Materials and Reagents

HEK293 cells
Poly-dL-Ornithine (PLO) (Sigma-Aldrich, catalog number: P8638 )
Boric Acid (Sigma-Aldrich, catalog number: B0394 )
Sulfo-NHS-SS-biotin solution (Thermo Fisher Scientific, Pierce Antibodies, catalog number: 21331 )
Neuroavidin beads (Thermo Fisher Scientific, Pierce Antibodies, catalog number: 29200 )
EDTA•2H₂O (MW 372.2) (Sigma-Aldrich, catalog number: E1644 )
EGTA•2H₂O (MW 380.4) (Sigma-Aldrich, catalog number: E4378 )
NaPyrophophate (Sigma-Aldrich, catalog number: S9515 )
NaF (Sigma-Aldrich, catalog number: S7920 )
Na₃VO₄•10H₂O (MW 446.1) (Sigma-Aldrich, catalog number: 5-9515 )
Protease inhibitor (F. Hoffmann-La Roche, catalog number: 1873580 )
NaVO₃
NaOH
PBS
CaCl₂
MgCl₂
Glycine
Triton
HCl
4x loading buffer
Biotin quenching solution (see Recipes)
PBS/CaCl₂/MgCl₂ (see Recipes)
IP buffer (see Recipes)
Stock (see Recipes)

Equipment

0.22 μm filters
12-well plates
Western blot equipment

Procedure

Prepare PLO solution: Add 90 mg PLO into 180 ml borate buffer (dissolve 1.668 g borate acid in 180 ml H₂O and adjust pH to 8.3 using NaOH), and sterilize by filtering through 0.22 μm filters.
Coat 12-well plates with PLO solution at room temperature overnight in the culture hood (1 ml per well). Wash three times with sterile water on the second day before cell plating.
Plate HEK293 cells on poly-ornithine coated 12-well plates and transfect as needed.
On the day of cell harvest, dissolve Sulfo-NHS-SS-Biotin in PBS/CaCl₂/MgCl₂ at 0.5 mg ml^-1 and store on ice.
Wash cells with 1 ml ice-cold PBS/CaCl₂/MgCl₂ twice.
Biotinylation
1. Add 0.3 ml of 0.5 mg/ml Sulfo-NHS-SS-biotin solution.
2. Incubate on ice for 30 min, with occasional shaking.
Wash three times with ice-cold quenching solution and incubate 5 min before each solution removal to allow neutralization of uncrosslinked reagents.
Lyse cells with 120 μl IP buffer and incubate 10 min on ice.
Transfer all contents of each well into a 1.5-ml tube and spin at Vmax (about 14,000 rpm) on a 4 °C desktop centrifuge for 10 min.
Save 10 μl supernatant (added 10 μl 4x loading buffer) as offers.
Mix 100 μl supernatants with 50 μl 50% slurry of immobilized Neutravidin and rotate for 2-3 h at 4 °C.
Spin 1 min at 5,000 rpm in a 4 °C centrifuge. Remove the supernatant and wash the beads with 1 ml IP buffer by rotating the mixture for 5 min in the 4 °C room. Wash three times in total.
After the final wash, remove the supernatant and add 25 μl 4x loading buffer to the beads. Mix well and put the tubes in a 65 °C water bath for 10 min to denature proteins (65 °C is chosen because we find that in boiling water, many membrane proteins form aggregates, which results in failure of proteins to migrate into acrylamide gels). Load 10 μl for western blot analysis.

Recipes

Biotin quenching solution (pH 7.4)
50 mM glycine (0.1877 g) in 50 ml PBS/CaCl₂/MgCl₂
PBS/CaCl₂/MgCl₂
PBS (+2.5 mM CaCl₂, 1 mM MgCl₂, pH 7.4)
IP buffer (pH 7.4)
PBS

50 ml
EDTA
5 mM
0.5 ml x 0.5 M (pH 7.4)
EGTA
5 mM
0.5 ml x 0.5 M (pH 7.4)
NaPyrophophate
10 mM
0.223 g
NaF
50 mM
0.1 g
NaVO₃
1 mM
0.5 ml x 100 mM
1% Triton
1 tablet Protease inhibitor
Stock
1. 0.5 M EDTA (pH 7.4)
  93.05 g EDTA•2H₂O
  500 ml ddH₂O
  Add NaOH until EDTA dissolves and add HCl to adjust pH back to 7.4.
2. 0.5 M EGTA (pH 7.4)
  95.1 g EGTA•2H₂O
  500 ml ddH₂O
  Add NaOH until EDTA dissolves and add HCl to adjust pH back to 7.4.
3. 100 mM Na₃VO₄
  1.84 g Na₃VO₄•10 H₂O
  Add HCl to pH less than 10 (yellow solution). Boil the solution until clear and colorless.
4. 10 ml aliquots and freeze at -20 °C.

Acknowledgments

This work was supported by National Institute on Drug Abuse (NIDA; DA00266, DA10309) and the National Institute of Mental Health (NIMH; MH068830). This protocol was previously used in and adapted from Huang et al. (2006).

References

Huang, G. N., Zeng, W., Kim, J. Y., Yuan, J. P., Han, L., Muallem, S. and Worley, P. F. (2006). STIM1 carboxyl-terminus activates native SOC, I(crac) and TRPC1 channels. Nat Cell Biol 8(9): 1003-1010.