Sensitive detection of metallothioneins-1, -2 and -3 in tissue homogenates by immunoblotting: A method for enhanced membrane transfer and retention

Standard immunoblotting procedures were unable to detect metallothioneins-1 and 2 (MT-1, MT-2) and metallothionein-3 (MT-3)/growth inhibitory factor (GIF) in unfractionated brain homogenates. We have developed a novel process which involves the inclusion of 2 mM CaCl2 in electrophoretic transfer buffers and glutaraldehyde fixation following transfer to either nitrocellulose or polyvinylidene difluoride (PVDF) membranes. Using commercial MT antibodies and a specific MT-3 polyclonal antibody raised in our laboratory, we have been able to detect all three MTs on both membrane types with a detection limit of approx. 10 ng for MT-1 and MT-2. Nitrocellulose membrane pore size had no noticeable effect on detection sensitivity. These modifications enable more sensitive MT detection than previously described blotting methods. In addition, this technique eliminates the need for indirect monitoring approaches and simplifies quantification since sample fractionation or enrichment are not required.