Determination of Protein Concentration

 Total membrane protein concentrations were determined by the bicinchoninic acid

(BCA) assay (286). The membrane preparations were diluted 1:10 for this assay. For

each sample, triplicates of 10 CIL, 20 CIL and sometimes 30 C1L of the diluted samples

were aliquoted for the assay. Bovine serum albumin (BSA) was used to generate a

standard curve. A stock of 1 mg/mL BSA was prepared and stored in microcentrifuge

tubes at -20 oC. The exact BSA concentration was determined from the OD280 (1.0

mg/mL BSA OD280=0.667). The BSA was aliquoted in triplicates ranging from 0 to 100

Clg (0, 5, 10, 20, 40, 60, 80, and 100 CIL) of protein. All of the samples were incubated in

3 mL of standard working reagent (SWR), consisting of 50 parts solution A (1% BCA-

Na2, 2% Na2CO3-H20, 0. 16% sodium potassium tartrate, 0.95% NaHCO3, pH 11.25 and

filtered) to one part solution B (4 % CuSO4-5H20) plus 1% sodium dodecyl sulfate

(SDS) mixed fresh as needed, for 30 minutes at 37 oC and then for 10 minutes at room

temperature. The absorbance at 562 nm was recorded for each sample with an LKB

Biochrom Ultrospec II spectrophotometer. The standard curve was generated from the

BSA OD readings, with a typical correlation coefficient of at least 0.997, and then protein

concentrations were determined using linear regression. A typical concentration range of

the total membrane protein prepared from 500 mL of media was 5-20 Clg/CIL.

Ni-Resin Purification

 Ni-resin purification was achieved using the High Capacity Nickel Chelate Affinity

Matrix (Ni-CAM) purchased from Sigma. It became necessary to optimize conditions

and the ratio of protein to packed Ni-resin volume for each type of experiment.

Determining the optimal ratio of protein to Ni-resin was critical because F1Fo ATP