Coomassie brilliant blue kit

The properties of the calcium-binding activity were investigated using the recombinant EFCAB2 protein, which covered the full length of the deduced EFCAB2 sequence except for the N-terminal six amino acids (MAEER), via Stains-all and ruthenium red. For Stains-all, first, the purified recombinant EFCAB2 protein was resolved by 12.5% SDS-PAGE, and the gels were stained with a Coomassie Brilliant Blue (CBB) kit (Nacalai Tesque, Japan); alternatively, the gels were washed overnight with 30% isopropyl alcohol, stained with Stains-all dye (Sigma) for 48 hours, and destained with isopropyl alcohol until a clear background was achieved. Second, the purified recombinant EFCAB2 protein was incubated with 5 μM Stains-all in 30% (v/v) ethylene glycol and 2 mM MOPS-KOH, pH 7.2, in the dark for 30 min, and the absorption spectra were obtained with a spectrophotometer as previously described [28 (link)]. For ruthenium red staining, the purified recombinant EFCAB2 protein was resolved by 12.5% SDS-PAGE, transferred to a PVDF membrane, and stained by Ponceau S or ruthenium red (25 mg/l of ruthenium red in 60 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl, pH 7.5) for 48 hours [29 (link)].