Context-dependent monoclonal antibodies against protein carbamidomethyl-cysteine

Abstract

Protein sulfhydryl residues participate in key structural and biochemical functions. Alterations in sulfhydryl status, regulated by either reversible redox reactions or by permanent covalent capping, may be challenging to identify. To advance the detection of protein sulfhydryl groups, we describe the production of new Rabbit monoclonal antibodies that react with carbamidomethyl-cysteine (CAM-cys), a product of iodoacetamide (IAM) labeling of protein sulfhydryl residues. These antibodies bind to proteins labeled with IAM (but not N-ethylmaleimide (NEM) or acrylamide) and identify multiple protein bands when applied to Western blots of cell lysates treated with IAM. The monoclonal antibodies label a subset of CAM-cys modified peptide sequences and purified proteins (human von Willebrand Factor (gene:vWF), Jagged 1 (gene:JAG1), Laminin subunit alpha 2 (gene:LAMA2), Thrombospondin-2 (gene:TSP2), and Collagen IV (gene:COL4)) but do not recognize specific proteins such as Bovine serum albumin (gene:BSA) and human Thrombospondin-1 (gene:TSP1), Biglycan (gene:BGN) and Decorin (gene:DCN). Scanning mutants of the peptide sequence used to generate the CAM-cys antibodies elucidated residues required for context dependent reactivity. In addition to recognition of in vitro labeled proteins, the antibodies were used to identify selected sulfhydryl-containing proteins from living cells that were pulse labeled with IAM. Further development of novel CAM-cys monoclonal antibodies in conjunction with other biochemical tools may complement current methods for sulfhydryl detection within specific proteins. Moreover, CAM-cys reactive reagents may be useful when there is a need to label subpopulations of proteins.

Hydrolysis of a second Asp-Pro site at the N-terminus of NOTCH3 in inherited vascular dementia

“…The 83G, 120B, and 145H rabbit monoclonal antibodies were generated for this study by standard procedures which are detailed elsewhere 24 . GenScript performed all animal experiments which were reviewed by the GenScript IACUC (animal protocol ANT19-005) and were performed in accordance with ARRIVE guidelines (a control group of non-peptide immunized animals was not included as this in not required for antibody generating procedures).…”

“…Human HEK293 cells grown to at least 90% confluence were transiently transfected using PolyJet (SignaGen, cat# SL100688) using to the manufacturer's recommended protocol 24 . NOTCH3-encoding constructs were mixed with vectors that encode puromycin resistance, and the resulting pool of cells was selected for resistance by supplementing growth media with the antibiotic; this process generally resulted in dozens of cell colonies which were individually selected and independently propagated.…”

“…We have previously described these methods 6 , 24 . Proteins were separated on SDS polyacrylamide gels, transferred to nitrocellulose with an iBlot 2 instrument (Invitrogen, method P0 20 V for 1 min/23 V for 4 min/25 V for 2 min), blocked in TBST supplemented with 5% milk, and then incubated with primary antibodies in TBST overnight at 4 °C.…”

Trans-Reduction of Cerebral Small Vessel Disease Proteins by Notch-Derived EGF-like Sequences

“…Three thiol detection methods were used: N-ethylmaleimide reaction of thiol groups followed by antibody-mediated detection of alkylation products [ 23 ] ( Figure 1 A); iodoacetamide reaction of thiol groups followed by antibody-mediated detection of alkylation products [ 24 ] ( Figure 1 B); and direct labeling of thiols with maleimide dye [Young KZ, accepted for publication] ( Figure 1 C). Each of the approaches demonstrated consistent increases in thiol alkylation of Fc-E3 protein after exposure to NTF2 (increased labeling in fourth lane compared to the third).…”

“…Protein samples for immunoblotting were prepared in sample buffer containing beta-mercaptoethanol and boiled. All samples were separated on standard 10% or 4–20% SDS-PAGE gradient gels (ThermoFisher, Waltham, MA, USA); after protein separation, electroblotting to nitrocellulose was performed using the iBlot 2 system [ 24 ]. Immunoblot analysis was performed with antibodies as indicated, followed by detection of primary probes using infrared fluorophore-labeled secondary antibodies (Rockland, Limerick, PA, USA).…”