Immunoblotting conditions for human hemoglobin chains

Immunoblotting to analyze low-molecular-weight proteins like calmodulin and metallothioneins is challenging and requires modifications for reproducible detection. Human globin chains are 17-kDa proteins and are not detectable by conventional immunoblotting using nitrocellulose membranes. Here we describe an immunoblotting method using nitrocellulose membranes that allows quantitative analyses of globin chains. Although previous studies have demonstrated that the fixation of blotted membranes with glutaraldehyde improves immunodetection of low-molecular-weight proteins, we found that the detection sensitivity for human globins is increased markedly by fixation with paraformaldehyde, but not glutaraldehyde. This immunoblotting procedure facilitates studies of posttranscriptional mechanisms for globin gene expression.