As described by Smith, the original BCA assay is a two-component protocol. The two reagents are "stable indefinitely at room temperature". Modern (likely exact or highly similar) formulations are available from at least two commercial vendors. The BCA Working solution is generated by mixing Reagent A and Reagent B in a 50:1 ratio, and can be prepared either weekly (it is moderately stable), or as needed.
Reagent A
* 1% w/v BCA-Na (CAS: 979-88-4)
* 2% w/v NaCO·HO (CAS: 5968-11-6)
* 0.16% w/v Na tartrate (CAS: 868-18-8)
* 0.4% w/v NaOH (CAS: 1310-73-2)
* 0.95% w/v NaHCO (CAS: 144-55-8)
* Add 50% NaOH or solid NaHCO to adjust the pH to 11.25
A suggested but untested alternative formulation in the Smith manuscript is to leave out the NaOH (and presumably not perform the manual pH adjustment to 11.25), but instead to dissolve the other components in a preprepared buffer of 0.25 M NaCO and 0.01 M NaHCO.
Notably, Smith synthesized their own BCA via the Pfitzinger reaction of isatin and acetoin, substituting NaOH for KOH but otherwise following the synthetic method of Lesene and Henze, as the BCA available from commercial vendors of that time was too impure for their use. At least three successive recrystallizations of their synthesized BCA from 70˚C water was needed to sufficiently purify it for the assay.
Reagent B
* 4% w/v CuSO·5HO