Wil T. Labruyère

Lack of specificity of commercially available antisera against muscarinergic and adrenergic receptors

Commercially available antisera against five subtypes of muscarinic receptors and nine subtypes of adrenoceptors showed highly distinct immunohistochemical staining patterns in rat ureter and stomach. However, using the M1–4 muscarinic receptor subtypes and α2B-, β2-, and β3-adrenoceptors as examples, Western blots with membranes prepared from cell lines stably expressing various subtypes of muscarinic receptors or adrenoceptors revealed that each of the antisera recognized a set of proteins that differed between the cell lines used but lacked specificity for the claimed target receptor. We propose that receptor antibodies need better validation before they can reliably be used.

Lack of Specificity of Commercially Available Antisera: Better Specifications Needed

The ideal antiserum for immunohistochemical (IHC) applications contains mono-specific high-affinity antibodies with little nonspecific adherence to sections. Many commercially available antibodies are “affinity” purified, but it is unknown if they meet “hard” specificity criteria, such as absence of staining in tissues genetically deficient for the antigen or a staining pattern that is identical to that of an antibody raised against a different epitope on the same protein. Reviewers, therefore, often require additional characterization. Although the affinity-purified antibodies used in our study on the distribution of muscarinic receptors produced selective staining patterns on sections, few passed the preabsorption test, and none produced bands of the anticipated size on Western blots. More importantly, none showed a difference in staining pattern on sections or Western blots between wild-type and knockout mice. Because these antibodies were used in most studies published thus far, our findings cast doubts on the validity of the extant body of morphological knowledge of the whole family of muscarinic receptors. We formulate requirements that antibody-specification data sheets should meet and propose that journals for which IHC is a core technique facilitate consumer rating of antibodies. “Certified” antibodies could avoid fruitless and costly validation assays and should become the standard of commercial suppliers.

Effect of arginine deficiency on arginine-dependent post-translational protein modifications in mice

Transgenic mice that overexpress arginase-I in their small-intestinal enterocytes suffer from a pronounced, but selective decrease in circulating arginine levels during the suckling period, resulting in impaired growth and development of hair, muscle and immune system. In the present study, we tested the hypothesis that the arginine-deficiency phenotype is caused by arginine-specific post-translational modifications, namely, an increase in the degree of mono-ADP-ribosylation of proteins because of reduced competition by free arginine residues and/or an increase in protein-tyrosine nitration because of an increased O2- production by NO synthases in the presence of limiting amounts of arginine. Arginine ADP-ribosylation and tyrosine nitration of proteins in the affected organs were assayed by Western blot analysis, using specific anti-ADP-ribosylarginine and protein-nitrotyrosine antisera. The composition of the group of proteins that were preferentially arginine ADP-ribosylated or tyrosine-nitrated in the respective organs was strikingly similar. Arginine-deficient mice differed from their controls in a reduced ADP-ribosylation of a 130 kDa and a 65 kDa protein in skin and an increased protein nitration of an 83 kDa protein in bone marrow and a 250 kDa protein in spleen. Since only 20 % of the visualised proteins were differentially modified in a subset of the affected organs, our findings appear to rule out these prominent arginine-dependent post-translational protein modifications as mediators of the characteristic phenotype of severely arginine-deficient mice.