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CYS1 binds CALM1. 30 / 30
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"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."
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"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)."
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"Scanning mutants of the peptide sequence used to generate the CAM-cys antibodies elucidated residues required for context dependent reactivity."
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"Further development of novel CAM-cys monoclonal antibodies in conjunction with other biochemical tools may complement current methods for sulfhydryl detection within specific proteins."
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"Moreover, CAM-cys reactive reagents may be useful when there is a need to label subpopulations of proteins."
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"To enhance the potential for antibodies to bind to reduced protein on immunoblots, a peptide corresponding to this sequence (CFDPKYGEPKRPPNCARGSCPWDSQL; accession # NP_001138585.2) was generated with a reduced N-terminal anchoring cysteine and two CAM-cys residues in place of the remaining native cysteines."
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"Of the antibodies identified by ELISA, we found by dot blotting that two rabbit IgG monoclonals (4E7 and 52H11) bound to the CAM-cys modified MYADML2 peptides but were incapable of binding to peptides lacking CAM-cys modifications (peptide sequences shown in xref ; blots shown in xref [probed with 4E7],1C [with 52H11], and 1D [with secondary antibody alone])."
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"We suspected that these MYADML2 non-reactive clones may, in fact produce, antibodies against CAM-cys that were incorporated in the immunizing peptide."
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"Breadth of proteins detected by CAM-cys monoclonal antibodies."
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"The parameters for the database search were tryptic peptides with one missed cleavage, carbamidomethylation (cys-CAM) as fixed modification, and oxidation (methionine) as variable modification, peptid[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"Both 4E7 and 52H11 detect ladders of protein on Western blots of populations of IAM treated cell lysates, suggesting that these affinity probes may recognize CAM-cys in multiple different proteins."
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"To distinguish whether the antibodies recognize CAM-cys from all proteins versus selected proteins, we tested whether the antibodies were capable of binding to specific purified proteins after IAM-treatment."
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"Proteins were then analyzed by Western blotting with 4E7 and 52H11. xref shows that antibodies against CAM-cys specifically reacted with proteins treated with IAM only after treatment with TCEP (compare first and second lanes of each group)."
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"Casein, which does not contain cysteines, was not recognized by OX133 or CAM-cys antibodies under any conditions."
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"These studies demonstrate the specificity and relative selectivity of CAM-cys antibodies for specific IAM-modified proteins."
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"Sequence specificity of CAM-cys monoclonal antibodies."
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"Searches were performed with 1-missed cleavage allowed, carbamidomethylcysteine as fixed modification, pyro-GLU with N-terminal glutamine, pyro-carbamidomethylcysteine with N-terminal CAM-CYS, methion[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
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"The antibodies to CAM-cys therefore appear to be very sensitive to single amino acid changes in the context of Western blotting."
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"This analysis reinforces that the monoclonal reagents recognize CAM-cys residues within specific contexts."
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"It should be noted that a peptide with two synthetic CAM-cys residues was recognized by antibodies on dot blots, which was different from Western blot studies (lane W, with IAM, xref )."
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"We describe the production of two monoclonal antibodies, 4E7 and 52H11, that target protein CAM-cys residues, a product of thiol labeling with IAM."
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"However, the target range of 4E7 and 52H11 differ from OX133 in that the two CAM-cys reagents appear to react with only a subset of protein sulfhydryl residues."
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"Multiple lines of evidence suggest that 4E7 and 52H11 are selective for a subset of CAM-cys residues."
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"Only one of two potential CAM-cys residues was absolutely essential to binding; and, in fact, when both cysteine residues were converted to CAM-cys, there was elimination of binding on Western blots."
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"Additional dot blot studies with synthetic peptides revealed the important residues adjacent to the first CAM-cys regulated binding, which further demonstrates the importance of sequence context."
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"To provide initial insight into the sequence specificity of the new CAM-cys antibodies, we annotated the primary sequences surrounding all cysteines of proteins that did not contain any residues that reacted on Western blots (S3 Fig in xref )."
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"Scanning peptide mutants ( xref ; first four sequences), demonstrated an increase in binding to CAM-cys peptides when an aspartic acid was changed to alanine and when an asparagine was mutated to alanine."
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"Thus, though the current study presents some information on the context that dictates binding preferences, definitive binding rules could not be determined, and binding to CAM-cys residues in other proteins is best determined experimentally."
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"Though the antibodies were capable of enriching for CAM-cys containing proteins, we found an unacceptable amount of background adherence of CAM-cys proteins to beads which limited the utility of the reagents for this application under the conditions tested (S4 Fig in xref )."
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"In the future, new antibodies developed against distinct CAM-cys antigens or independent sulfhydryl-reactive probes [ xref – xref ] could be used for discrimination of sulfhydryl status of specific sequences."
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