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Prices are in US dollars and valid only for US orders.
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Description:
The Peptide-Carrier Kit™ allows peptides of interest to be detected on Western blots or peptide arrays through ligation to a carrier protein. The resulting peptide-carrier ligation product is compatible with a variety of applications including Western blotting, peptide arrays and kinase assays (1-3). Simply mixing a peptide (N-terminal cysteine required) with the supplied Carrier Protein 39 (CP39, ~39 kDa) results in a peptide-carrier ligation product via intein-mediated protein ligation (IPL, 4-6).
Since most antigenic peptides are routinely synthesized with a cysteine, a single peptide containing an N-terminal cysteine can now be used to generate antibodies as well as a control for Western blots. The antigenic peptide may contain modified residues, such as a phosphorylated tyrosine. The ligated product has one antigenic peptide per CP39 resulting in a single, sharp band on Western blots (Figure 1)(2).
In addition, peptide substrates ligated to Carrier Proteins 39 (NEB #E6602) or 27 (NEB #E6606) can be used for enzymatic assays and subsequent Western blot analysis (Figure 3) (3). This kit also makes it possible to produce peptide substrates for enhanced sensitivity in array analysis (Figures 4 & 5) (1).
Figure 1: Western blot analysis of antiserum raised against Control Peptide, PB1. PB1 was ligated to Carrier Protein 39 (Western) (NEB #E6602) as in Protocol I on the Peptide-Carrier Kit (NEB #E6600) 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. 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 Peptide-Carrier Kit (NEB #E6600) 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).
Figure 3: Kinase Assay. A peptide containing a candidate phosphorylation site was synthesized with an additional N-terminal cysteine (CGSNEAIYAAPFAKKK; 7) and ligated to Carrier Protein 39 (Western). The ligated product was phosphorylated with Abl Protein Tyrosine Kinase (NEB #P6050) and then subjected to Western blot analysis using anti phospho tyrosine antibody (Cell Signaling Technology). A positive signal was detected only in lanes 3 and 4 where the ligated product was treated with Abl Kinase.
Lane 1: Carrier Protein 39 (Western), CP39
Lane 2: CP39 + Abl Kinase (100 units)
Lane 3: CP39-peptide + Abl Kinase (50 units)
Lane 4: CP39-peptide + Abl Kinase (100 units)
Lane 5: CP39-peptide
Lane 6: Abl Kinase (100 units)
Figure 4: Comparison of CP39-PB1 and unligated PB1 peptide by dot blot analysis on a nitrocellulose membrane.
Figure 5A: 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 5B: Alanine scan of the hemagglutinin (HA) epitope.
The ligated products (rows 1-4) were arrayed on a 0.45 µm nitrocellulose membrane along with unligated peptides (rows 5-8). Each peptide was ligated to Carrier Protein 39 (Array) (NEB #E6603) overnight at 4°C. The blot was reacted with an anti-HA monoclonal antibody.
Applications:- Generation of positive controls for Western blot analysis (2)
- Production of substrates for enhanced sensitivity in peptide array or dot blot assay (1)
- Production of substrates for protein modification, such as phosphorylation by kinase(3)
- Ligation of a peptide containing natural or unnatural amino acids
Kit Components:
Carrier Protein (CP) Reaction Buffer (10X)
Carrier Protein 39 (Western)
Control Peptide, PB1 (2X)
SDS Sample Buffer (3X)
Storage Conditions
Storage Temperature:
-70°C
Avoid repeated freeze/thaw cycles.
Notes
General notes:- Can be stored for 1 week or less at -20°C.
FAQs
- How is the peptide ligated to the carrier protein?
- Do I have to run a Coomassie stained SDS-PAGE gel before the Western blot?
- What are the causes of inefficient ligation?
- Is Western blot analysis possible if the extent of ligation is not very good?
- What controls can be done for a Western blot?
- Can an insoluble peptide be used?
- Does the peptide have to be purified?
- How do I calculate the concentration of the peptide solution?
- Can a ligated peptide be used for Enzyme Linked Immunosorbent Assays (ELISA)?
- Can I use crude sera in the Western blot analysis?
- What is the difference between CP27 and CP39?
- When do I need to use CP27?
- What is the difference between CP39 (Western) and CP39 (Array)?
- What is the shelf life of the carrier protein (CP)?
Appendix: Chemical Mechanism of Intein Mediated Protein ligation (IPL)
The IPL reaction allows the ligation of a synthetic peptide with an N-terminal cysteine residue to a bacterially expressed protein with a C-terminal thioester through a native peptide bond (4,6). To create a C-terminal thioester the IMPACT-TWIN System (NEB #E6950S) may be used (4,6).
References
- Sun, L., Rush, J., Ghosh, I., Maunus, J. 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.
- 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.
- Songyang, Z., Carraway, K.L. 3rd, Eck, M.J. Harrison, R.A. Feldman, R.A. Mohammadi, M., Schlessinger, J., Hubbard, S.R. Smith, D.P. Eng, C. Lorenzon, J. Ponder, B.A. J. Mayer, B.J. and Cantley, L.C. (1995) Nature, 373, 536-539.
Reagents Sold Separately
Carrier Protein (CP) Reaction Buffer
Carrier Protein 39 (Western)
Control Peptide, PB1
Companion Products
Anti-PB1 Peptide Antibody
Bio-P1 (Lyophilized)
Carrier Protein (CP) Reaction Buffer
Carrier Protein 27 (Array)
Carrier Protein 27 (Western)
Carrier Protein 39 (Array)
Carrier Protein 39 (Western)
Control Peptide, PB1
Flu-P1 (Lyophilized)
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