Domain unfolding of monoclonal antibody fragments revealed by non-reducing SDS-PAGE

Investigation of monoclonal antibody fragmentation artifacts in non-reducing SDS-PAGE.

Fragmentation of monoclonal antibodies has been routinely observed in non-reducing SDS-PAGE, mainly due to disulfide-bond scrambling catalyzed by free sulfhydryl groups, resulting in a method induced artifact. To minimize this artifact, alkylating agents like iodoacetamide (IAM) and N-ethylmaleimide (NEM) were commonly included in SDS sample buffer to block free sulfhydryls. However, the select...

Different mechanisms of antibody-mediated neutralization of parvoviruses revealed using the Fab fragments of monoclonal antibodies.

Antibody binding and neutralization are major host defenses against viruses, yet the mechanisms are often not well understood. Eight monoclonal antibodies and their Fab fragments were tested for neutralization of canine parvovirus and feline panleukopenia virus. All IgGs neutralized >85% of virus infectivity. Two Fabs neutralized when present at 5 nM, while the others gave little or no neutrali...

Domain interactions in antibody Fv and scFv fragments: effects on unfolding kinetics and equilibria.

The equilibrium denaturation and unfolding kinetics of the domains V(L) and V(H) have been compared with those of the Fv and single-chain Fv (scFv) fragment of an engineered variant of the antibody McPC603 in the presence and absence of the antigen phosphorylcholine. The scFv fragment is significantly more stable than the isolated constituting domains. Antigen binding stabilizes the heterodimer...

Tricine – SDS - PAGE Hermann Schägger

Tricine–SDS-PAGE is commonly used to separate proteins in the mass range 1–100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. The concentrations of acrylamide used in the gels are lower than in other electrophoretic systems. These lower concentrations facilitate electroblotting, which is particularly crucial for hydrophobic proteins. Tricine–...

Identification of electrophoretic proteins pattern of Fusaium verticillioides isolates from maize by SDS-PAGE.