IndraLab

Statements

CALM1 binds CYS1. 28 / 28
| 28
sparser
"This analysis reinforces that the monoclonal reagents recognize CAM-cys residues within specific contexts."
33232360
sparser
"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."
33232360
sparser
"These studies demonstrate the specificity and relative selectivity of CAM-cys antibodies for specific IAM-modified proteins."
33232360
sparser
"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)."
33232360
sparser
"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])."
33232360
sparser
"Breadth of proteins detected by CAM-cys monoclonal antibodies."
33232360
sparser
"Multiple lines of evidence suggest that 4E7 and 52H11 are selective for a subset of CAM-cys residues."
33232360
sparser
"Casein, which does not contain cysteines, was not recognized by OX133 or CAM-cys antibodies under any conditions."
33232360
sparser
"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 )."
33232360
sparser
"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."
33232360
sparser
"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."
33232360
sparser
"Scanning mutants of the peptide sequence used to generate the CAM-cys antibodies elucidated residues required for context dependent reactivity."
33232360
sparser
"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."
33232360
sparser
"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 )."
33232360
sparser
"We suspected that these MYADML2 non-reactive clones may, in fact produce, antibodies against CAM-cys that were incorporated in the immunizing peptide."
33232360
sparser
"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."
33232360
sparser
"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."
33232360
sparser
"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."
33232360
sparser
"Moreover, CAM-cys reactive reagents may be useful when there is a need to label subpopulations of proteins."
33232360
sparser
"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."
33232360
sparser
"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 )."
33232360
sparser
"The antibodies to CAM-cys therefore appear to be very sensitive to single amino acid changes in the context of Western blotting."
33232360
sparser
"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)."
33232360
sparser
"We describe the production of two monoclonal antibodies, 4E7 and 52H11, that target protein CAM-cys residues, a product of thiol labeling with IAM."
33232360
sparser
"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."
33232360
sparser
"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."
33232360
sparser
"Further development of novel CAM-cys monoclonal antibodies in conjunction with other biochemical tools may complement current methods for sulfhydryl detection within specific proteins."
33232360
sparser
"Sequence specificity of CAM-cys monoclonal antibodies."
33232360