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Carrier Protein 27 (Array) |
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Description:
Carrier Protein 27 (Array) is a ~27 kDa protein possessing a C-terminal thioester to which a peptide with a N-terminal cysteine can be ligated (1-3). This procedure is termed intein mediated protein ligation (IPL; 4-6). The ligated product can be used in peptide arrays and western blots (1; Refer to the Peptide-Carrier™ Kit Instruction Manual, NEB #E6600).
Peptide Array or Dot Blot Assay:
This versatile method has made it possible to produce peptide substrates for array analysis in research laboratories (1). Carrier Protein 27 (Array) is utilized as a carrier to improve the binding of peptide substrates to commonly used membranes (such as nitrocellulose, nylon and PVDF). Increased peptide binding significantly enhances the sensitivity of detection in antibody binding assays, such as epitope mapping. Since the ligation stoichiometry is precisely one-to-one, synthetic peptides ligated to a carrier protein become normalized during array production.
Figure 1: Immunoblotting was performed with Anti-PB1 Peptide Antibody (NEB #E6608).
Figure 2A: Alanine scan of the hemagglutinin (HA) epitope
HA peptide library was synthesized with an N-terminal cysteine. P9 contains the wild type sequence corresponding to residues Tyr98 to Ala106 of the hemagglutinin (HA) protein. Each of the other 8 peptides (P1-P8) carries a single substitution with an alanine residue.
Figure 2B: Alanine scan of the hemagglutinin (HA) epitope
The ligated products (rows 1-4) were arrayed on a 0.45 µm nitrocellulose membrane along with the unligated peptide (rows 5-8). Each peptide was ligated to Carrier Protein 27 (Array) (NEB #E6607) overnight at 4°C. The blot was reacted with an anti-HA monoclonal antibody (Zymed Laboratory).
Figure 3: Analysis of peptide ligation by Coomassie Blue stained SDS-PAGE. The Control Peptide, PB1, was ligated to Carrier Protein 27 (Array), as in Protocol II on the Carrier Protein 27 (Array) (NEB #E6607) datacard. The ligation samples were electrophoresed on a 12% SDS polyacrylamide gel and examined by Coomassie Blue staining. Lanes 1 and 5 are controls (C) which contain only Carrier Protein 27 (Array).
Source:
Carrier Protein 27 (Array) is a protein from Dirofiliaria immitis. The thioester tagged protein is expressed in E.coli and purified from the intein tag by cleaving with 2-mercaptoethanesulfonic acid (MESNA).
Reaction & Storage Conditions
Concentration:
1 mg/ml
Storage Conditions:
5 mM Bis-Tris
0.25 mM NaCl
pH 6.0 @ 25°C
Storage Temperature:
-70°C
Avoid repeated freeze/thaw cycles.
Notes
General notes:- It is not necessary to run Coomassie Blue stained SDS-PAGE unless you wish to check the extent of ligation. For enzymatic assays Protocol II on the Carrier Protein 27 (Array) (NEB #E6607) datacard can be used to produce the substrates: adjust substrate concentration as recommended. In this case do not add 3X SDS Sample Buffer.
- Can be stored for 1 week or less at -20°C. Should be stored at -70°C upon arrival.
Usage notes:- 75-90% of the Carrier Protein 27 (Array) is ligated when the purified peptide is used at a final peptide concentration of 0.5-1 mM. Use of certain unpurified peptides may result in lower ligation efficiency (10-50%). This will still result in a positive signal in array analysis.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Analysis:
Carrier Protein 27 (Array) is ligated to the Control Peptide PB1 (NEB #S6605) containing a N-terminal cysteine for 60 minutes at room temperature. A positive signal is detected on Coomassie Blue staining of a SDS-PAGE.
References
- Sun, L., Rush, J., Ghosh, I., Maunus, J.R. and Xu, M.-Q. (2004) Biotechniques, 37, 430-443.
- Ghosh, I., Sun, L., Evans., T.C., Jr. and Xu, M.-Q. (2004) J. Immunol. Methods, 293, 85-95.
- Xu, J., Sun, L., Ghosh, I. and Xu, M.-Q. (2004) Biotechniques, 36, 976-981.
- Evans, T.C., Jr., Benner, J. and Xu, M.-Q. (1998) Protein Sci., 7, 2256-2264.
- Evans, T.C., Jr. and Xu, M.-Q. (1999) Biopolymers, 51, 333-342.
- Muir, T.W., Sondhi, D. and Cole, P.A. (1998) Proc. Natl. Acad. Sci. USA, 95, 6705-6710.
Companion Products
Anti-PB1 Peptide Antibody
Carrier Protein (CP) Reaction Buffer
Carrier Protein 27 (Western)
Carrier Protein 39 (Array)
Carrier Protein 39 (Western)
Control Peptide, PB1
Peptide-Carrier™ Kit
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