chapter, cysteine chemical modifications were created in the 6 and b subunits to provide

reactive thiols groups for future labeling studies.

 Materials and Methods

 A more thorough account of many of the procedures used in Chapter 6 can be

found in previous chapters. Many of the techniques, including recombinant DNA

techniques, site directed mutagenesis, crude membrane preparation procedures, western

blotting, as well as assays of protein concentration and F1Fo ATP synthase activity have

been described in detail in Chapter 2.

Materials

 Molecular biology enzymes and mutagenic oligonucleotides were obtained from

Invitrogen (Carlsbad, CA), Life Technologies, Inc. (Grand Island, NY), New England

Biolabs (Beverly, MA) and Stratagene (La Jolla, CA). Reagents were obtained from

Sigma (St. Louis, MO), BioRad Laboratories (Hercules, CA) and Fisher Scientifie

(Pittsburgh, PA). Plasmid purification kits were acquired from Qiagen Inc. (Valencia,

CA). The anti-rabbit immunoglobulin horseradish peroxidase-linked whole antibody

(from donkey), Hybond ECL Nitrocellulose membrane, electrochemiluminescence

Western blotting reagents and high performance chemiluminescence fi1m were purchased

from Amersham Biosciences (Piscataway, NJ). Polyclonal antibodies against SDS-

denatured b subunit (284, 285) were generously provided by Dr. Karlheinz Altendorf

(Universitait Osnabruick, Osnabruick, Germany.

Strains and Media

 The bacterial strains used to create the cysteine chemical modifications in the b

subunit include the wild type b subunit expression plasmid, pKAM14 (b, Apr), and

plasmids used to express b subunits shortened or lengthened by 11 amino acids, pAUL5