Revision 3

#80353

Store at -20C

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Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Applications:
W, FC-FP

Reactivity:
All

Sensitivity:
Endogenous

Source/Isotype:
Rabbit IgG

Product Usage Information

Application Dilution
Western Blotting 1:1000
Flow Cytometry (Fixed/Permeabilized) 1:50 - 1:200

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity/Sensitivity

Carbamidomethyl Cysteine (F3T7J) Rabbit Monoclonal Antibody recognizes a broad range of carbamidomethylated cysteine containing proteins and peptides. This antibody does not cross-react with cysteines alkylated with desthiobiotin iodoacetamide (DBIA).

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide containing carbamidomethylated cysteine flanked by degenerate amino acids at positions N- and C-terminal to the modified cysteine.

Background

Cysteine alkylation is often performed with iodoacetamide (IAA) or chloroacetamide (CAA) to generate a carbamidomethyl (CAM) modification. Because alkylation occurs on reduced cysteines, disulfide-bonded cysteines must first be treated with dithiothreitol (DTT) or another reducing agent. The alkylation reaction is irreversible. Consequently, alkylated cysteines are unable to re-associate with each other. If the sulfhydryl group of a cysteine is already modified by another compound, that cysteine will not be able to react with IAA to form the carbamidomethyl modification. The alkylation reaction is rapid, although the efficiency can be influenced by the denaturant type used (1).

Proteins are often derivatized to prevent disulfide bond scrambling that can lead to artifactual bands in gel electrophoresis, and to minimize protease inaccessibility of cysteine-containing regions that can limit coverage in mass spectrometry-based proteomics. In light of various studies describing potential non-cysteine reactivities of IAA and CAA and re-evaluating the effectiveness of such reactions, users should consider the benefits of cysteine derivatizations depending on their specific experimental goals (2-4).

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

W: Western Blotting FC-FP: Flow Cytometry (Fixed/Permeabilized)

Cross-Reactivity Key

All: All Species Expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 3

Cell Signaling Technology Logo
Western blot analysis of extracts from HEK293 cells, untreated (0 mM; -) or spiked with 10% of iodoacetamide-treated (10 mM; +) extracts, using Carbamidomethylated Cysteine (F3T7J) Rabbit mAb or DJ-1 (D29E5) XP® Rabbit mAb #5933 and detected with Anti-rabbit IgG (H+L) (DyLight 680 Conjugate) #5366. The specific carbamidomethylated cysteine (CAM) signal (first panel) was blocked by the modified peptide (third panel), but not by the corresponding unmodified peptide (second panel).
Western Blotting Image 1: Carbamidomethyl Cysteine (F3T7J) Rabbit Monoclonal Antibody
Chemical structure of unmodified (left) and carbamidomethylated (right) cysteine generated with alkylating reagent.
Western Blotting Image 2: Carbamidomethyl Cysteine (F3T7J) Rabbit Monoclonal Antibody
Flow cytometric analysis of REC-1 cells, untreated (blue) or treated post-fixation with iodoacetamide (0.2 mM, 15 min; green), using Carbamidomethyl Cysteine (F3T7J) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Flow Cytometry Image 1: Carbamidomethyl Cysteine (F3T7J) Rabbit Monoclonal Antibody
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.