 |
|
 |
|
|
| Home >
Products >
Buffers >
Buffers and Supplements >
Carrier Protein (CP) Reaction Buffer |
 | | | | Carrier Protein (CP) Reaction Buffer | | | |
|
|
 |
|
Prices are in US dollars and valid only for US orders.
|
Description:
The 10X Carrier Protein (CP) Reaction Buffer is used at 1X concentration to ensure intein-mediated protein ligation (IPL; 1-6) between a thioester tagged carrier protein and an N-terminal cysteine containing peptide.
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 Carrier Protein Reaction Buffer (NEB #B6604) 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). 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).
Reagents Supplied:
Carrier Protein (CP) Reaction Buffer
1X Carrier Protein (CP) Reaction Buffer:
100 mM Tris-HCl
10 mM MESNA
pH 8.5 @ 4°C
Storage Conditions
Storage Temperature:
-20°C
Avoid repeated freeze/thaw cycles.
Notes
General notes:- 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.
- 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 CP Reaction Buffer (NEB #B6604) 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 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) (NEB #E6602) is ligated to the Control Peptide PB1 (NEB #S6605) containing an N-terminal cysteine (CTRSRHSSYPNEYEEDEEMEEEL; MW, 2862 Da) in the presence of 1X Carrier Protein Reaction Buffer for 60 minutes at room temperature. A positive signal is detected on Western blot analysis with Anti-PBI Antibody (1:2000) (NEB #E6608).
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.
Companion Products
Anti-PB1 Peptide Antibody
Carrier Protein 27 (Array)
Carrier Protein 27 (Western)
Carrier Protein 39 (Array)
Carrier Protein 39 (Western)
Control Peptide, PB1
Peptide-Carrier™ Kit
|
|
|
 |
|
|
