Manual C-terminal Protein Labeling Kit - Jena Bioscience

Manual
C-terminal Protein Labeling Kit
Universal C-terminal Protein Labeling with Coumarin
Protein Labeling
Cat.-No. Amount
FP-301-COU
For in vitro use only
Quality guaranteed for 12 months
Store at -20°C
Avoid freeze / thaw cycles
for labeling of
5 mg protein
Keto-Coumarin must be stored in the dark
Kit contents
pTWIN Vector (red cap)
5 µg modified pTWIN vector
Bisoxyamine (blue cap)
100 µl 1M in Reaction Buffer
MESNA (yellow cap)
35 mg (Sodium 2-mercaptoethanesulfonate)
Keto-Coumarin (purple cap)
12.5 µl 100 mM
Aniline
10 mg
Description
Coumarin Protein-C-labeling Kit provides a highly
efficient and easy-to-handle tool for the C-terminal
modification of proteins based on a chemoselective
oxime ligation. The rapid reaction of oxyamino
modified proteins with ketones under mild condition is
potentially beneficial for many biological applications.
The kit contains Keto-Coumarin as fluorescent label.
Background
Site-specific protein modification can facilitate the
characterization of protein functions both in
biochemical and in cellular investigations. Although
many chemical reactions are applicable in principle,
methods for the site-specific modification of proteins
remain in high demand, and there is a requirement for
readily available ligation reagents and mild reaction
conditions. Oxime-based reactions have found wide
application in the conjugation of biomolecules on
account of the absence of oxyamino groups in
proteins and their orthogonal reactivity with ketones to
give stable oximes.
The oxyamine–ketone bioorthogonal reaction has
been exploited in protein modification mainly by
means of incorporating ketone groups into proteins by
various chemical, enzymatic, and molecular
biological methods. To expand the application of this
efficient methodology to protein ligation, a simple and
general method to incorporate oxyamino groups into
proteins has been developed.
Structure
Bisoxyamine Keto-Coumarin
Spectroscopic data of coumarin
Excitation maximum: λEx = 332 nm
Emission maximum: λEm = 410 nm
Jena Bioscience GmbH | Löbstedter Str. 80 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100
http://www.jenabioscience.com Last update: Jun 03, 2009

Manual
C-terminal Protein Labeling Kit
Universal C-terminal Protein Labeling with Coumarin
Protein Labeling
Procedure
Protein Preparation with C-terminal-thioesther
1. Clone target gene into modified pTWIN vector
using NdeI and SapI sites.
2. Express fusion protein (target-Intein-His) in
BL21(DE3) cells.
3. Collect cells in 25 mL ice-cold Breaking Buffer
freshly supplemented with 1 mM PMSF.
CRITICAL: PMSF should be added freshly. Don’t
add any reducing substances.
4. Lyse cells using a microfluidizer or ultrasonication.
5. Add 1% Triton X-100 into cell lysate and
centrifuge at 35,000 rpm, 4°C for 30 min.
6. Filter supernatant through a 0.2 μm filter.
7. Load cell lysate onto a Ni-NTA column
equilibrated with Buffer A.
8. Wash column with Buffer A and continue with
2% Buffer B until absorbance reaches baseline.
9. Elute column with a gradient of 2-100% Buffer B.
Collect eluted fractions.
10. Identify and collect fractions of interest by SDS-
PAGE.
11. Add MESNA powder to protein solution to a
concentration of 0.5 M and incubate overnight at
20 °C.
12. Dilute solution with 5-fold volume of Buffer A
13. Load them onto a Ni-NTA column equilibrated
with Buffer A containing 10 mM MESNA.
Collect flow-through.
14. Wash column with 2-5% Buffer B containing 10
mM MESNA. Collect and pool flow-through and
concentrate protein.
15. Run a gel filtration on a Superdex column using
Elution Buffer.▲CRITICAL: Prepare fresh solution,
filter buffer through a 0.2 μm filter and degas on
a vacuum-membrane pump by stirring for 0.5 h at
room temperature.
16. Identify and collect fractions of interest by SDS-
PAGE. Concentrate protein and snap-freeze in
liquid nitrogen. Store protein at -80°C.
Protein C-terminal Labeling
17. Incubate 200 µl protein-thioester (5-25 mg/ml)
with 100 µl Bisoxyamine (1 M stock solution in
reaction buffer, final 333 mM) in Reaction
Buffer on ice overnight.
The reaction is monitored by ESI-MS.
18. Dialyze protein twice against 1 L Dialysis
Buffer at 4 °C.
19. Incubate 50 µM protein-ONH2 with 0.5 mM
Keto-Coumarin for 20 h or 1 mM Keto-
Coumarin overnight on ice in the presence of
100 mM Aniline in Incubation Buffer.
CRITICAL: If your protein can tolerate an acidic
environment, the reaction can also be performed
by incubating 50 µM protein-ONH2 with 1 mM
Keto-Coumarin for 4 h in NaAc Buffer.
20. Remove excess dye using a desalting column preequilibrated
in Dialysis Buffer.
Jena Bioscience GmbH | Löbstedter Str. 80 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100
http://www.jenabioscience.com Last update: Jun 03, 2009

Manual
C-terminal Protein Labeling Kit
Universal C-terminal Protein Labeling with Coumarin
Protein Labeling
To be provided
Breaking Buffer
25 mM NaH2PO4 pH 7.5, 0.5 M NaCl
PMSF
Triton X-100
Buffer A
50 mM NaH2PO4 pH 8.0, 0.3 M NaCl
Buffer B
50 mM NaH2PO4 pH 8.0, 0.3 M NaCl,
0.5 M imidazole
Elution Buffer
30 mM NaH2PO4 pH 7.5, 50 mM NaCl,
10 mM MESNA
Reaction Buffer
30 mM NaH2PO4 pH 7.5, 50 mM NaCl
Dialysis Buffer
30 mM NaH2PO4 pH 7.5, 50 mM NaCl, 2 mM DTE
Incubation Buffer
30 mM NaH2PO4 pH 7.0, 50 mM NaCl, 2 mM DTE
NaAc Buffer
50 mM NaAc pH 5.5, 50 mM NaCl, 2 mM DTE
Jena Bioscience GmbH | Löbstedter Str. 80 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100
http://www.jenabioscience.com Last update: Jun 03, 2009

Manual
C-terminal Protein Labeling Kit
Universal C-terminal Protein Labeling with Coumarin
Protein Labeling
Selected References
Yi, L. et al. (2010) A Highly Efficient Strategy for Modification of
Proteins at the C Terminus. Angewandte Chemie Int. Ed. 49:9417.
Yi, L. et.al. (2011) One-Pot Dual-Labeling of a Protein via Two
Chemoselective Reactions. Angewandte Chemie Int. Ed. 50: 8287.
Jena Bioscience GmbH | Löbstedter Str. 80 | 07749 Jena, Germany | Tel.:+49-3641-6285 000 | Fax:+49-3641-6285 100
http://www.jenabioscience.com Last update: Jun 03, 2009