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Carrier Protein 39 (Western) |
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Description:
Carrier Protein 39 (Western) is a ~39 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 Western blot analysis and kinase assays (Refer to the Peptide-Carrier™ Kit Manual, NEB #E6600).
Figure 1: Western blot analysis of antiserum raised against Control Peptide, PB1 (NEB #S6605). PB1 was synthesized with a N-terminal cysteine (CTRSRHSSYPNEYEEDEEMEEEL; MW, 2862 Da) and ligated to Carrier Protein 39 (Western) (NEB #E6602) as in Protocol I on the Carrier Protein 39 (Western) (NEB #E6602) datacard. The reaction times and temperatures are indicated. The samples were electrophoresed on a 12% SDS polyacrylamide gel and transferred to nitrocellulose. Anti-PB1 Peptide Antibody (NEB #E6608) was used as the primary antibody at 1:2000 and HRP-linked anti-rabbit antibody was used as the secondary antibody at 1:1500. Lanes 1 and 5 are controls (C) which contain only Carrier Protein 39 (Western).
Figure 2: Analysis of peptide ligation by Coomassie Blue stained SDS-PAGE. The Control Peptide, PB1, was ligated to Carrier Protein 39 (Western), as in Protocol II on the Carrier Protein 39 (Western) (NEB #E6602) 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 39 (Western).
Source:
Carrier Protein 39 (Western) is a protein from Haemophilus haemolyticus. 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:
0.2 mg/ml
Storage Conditions:
5 mM Bis-Tris
0.25 M 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.
- Can be stored for 1 week or less at -20°C. Should be stored at -70°C upon arrival.
- The sample can be stored at -20°C after the addition of 3X SDS Sample Buffer. For the control reaction use water instead of the Peptide Solution. For enzymatic assays Protocol II on the Carrier Protein 39 (Western) (NEB #E6602) datacard can be used to produce the substrates: adjust substrate concentration as recommended. In this case do not add 3X SDS Sample Buffer.
Usage notes:- 75-90% of the Carrier Protein 39 (Western) 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 on Western blot 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 39 (Western) is ligated to the Control Peptide PB1 (NEB #S6605) containing an N-terminal cysteine (CTRSRHSSYPNEYEEDEEMEEEL; MW, 2862 Da) for 60 minutes at room temperature. A positive signal is detected on Western blot analysis with Anti-PB1 Antibody (1:2000) (NEB #E6608).
References
- Sun, L., Rush, J., Ghosh, I., Maunus, J.R. and Xu, M.-Q. (2004) Biotechniques, 37, 730-443.
- 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.
- Muir, T.W., Sondhi, D. and Cole, P.A. (1998) Proc. Natl. Acad. Sci. USA, 95, 6705-6710.
- Evans, T.C., Jr. and Xu, M.-Q. (1999) Biopolymers, 51, 333-342.
Companion Products
Anti-PB1 Peptide Antibody
Carrier Protein (CP) Reaction Buffer
Carrier Protein 27 (Array)
Carrier Protein 27 (Western)
Carrier Protein 39 (Array)
Control Peptide, PB1
Peptide-Carrier™ Kit
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