Solid-phase metal chelate assay for quantifying total protein: resistance to chemical interference

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• Reversible metal chelate stains have been developed for protein analysis.
• These stains are compatible with immunoblotting and protein sequencing techniques.

Purpose of the Study:

• To evaluate metal chelate stains, specifically the double-metal chelate (DMC) stain, for routine protein quantitation.
• To compare the performance of Ferrozine/ferrous and DMC assays against colloidal gold and bicinchoninic acid (BCA) assays.

Main Methods:

• Proteins were quantified using computerized densitometry after membrane incubation with Ferrozine/ferrous complex and ferrocyanide/ferric complex (DMC stain).
• Samples were applied to membranes using a slot-blot apparatus for consistent application area.
• Assays were compared based on chemical interference, protein-to-protein variation, linear range, and sensitivity.

Main Results:

• The DMC assay demonstrated a superior linear range (100-fold) compared to BCA assays (47-fold).
• Colloidal gold assay showed higher sensitivity but suffered from poor reproducibility and high protein-to-protein variation.
• BCA assay exhibited the least protein-to-protein variation but was least sensitive and most susceptible to interfering agents.

Conclusions:

• Metal chelate stains, particularly the DMC assay, are effective for routine protein quantitation.
• The DMC assay offers a robust linear range and compatibility with downstream applications like immunoblotting and sequencing.