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C-terminal Protein Labeling Kit

Universal C-terminal Protein Labeling with Coumarin

Cat. No. Amount Price (EUR) Buy / Note
FP-301-COU 1 Kit (for labeling of 5 mg protein) 320,00 Add to Basket/Quote Add to Notepad

For in vitro use only!

Shipping: shipped on blue ice

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles, store dark

Shelf Life: 12 months

Spectroscopic Properties: λexc 332 nm; λem 410 nm (Keto-Coumarin)

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.

Kit contents:
pTWIN Vector (red cap)
5 μg modified pTWIN vector

Bisoxyamine (blue cap)
111 μl 0.9 M in Reaction Buffer

MESNA (yellow cap)
35 mg (Sodium 2-mercaptoethanesulfonate)

Keto-Coumarin (purple cap)
12.5 μl 100 mM

Aniline
10 mg

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

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.

Procedure:
Protein Preparation with C-terminal-thioesther

  • Clone target gene into modified pTWIN vector using Nde I and Sap I sites.
  • Express fusion protein (target-Intein-His) in BL21(DE3) cells.
  • 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.
  • Lyse cells using a microfluidizer or ultrasonication.
  • Add 1 % Triton X-100 into cell lysate and centrifuge at
    35,000 rpm, 4 °C for 30 min.
  • Filter supernatant through a 0.2 μm filter.
  • Load cell lysate onto a Ni-NTA column equilibrated with Buffer A.
  • Wash column with Buffer A and continue with 2 % Buffer B until absorbance reaches baseline.
  • Elute column with a gradient of 2-100 % Buffer B. Collect eluted fractions.
  • Identify and collect fractions of interest by SDS-PAGE.
  • Add MESNA powder to protein solution to a concentration of
    0.5 M and incubate overnight at 20 °C.
  • Dilute solution with 5-fold volume of Buffer A.
  • Load them onto a Ni-NTA column equilibrated with Buffer A containing 10 mM MESNA. Collect flow-through.
  • Wash column with 2-5 % Buffer B containing 10 mM MESNA. Collect and pool flow-through and concentrate protein.
  • 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.
  • 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

  • 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.
  • Dialyze protein twice against 1 l Dialysis Buffer at 4 °C.
  • 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.
  • Remove excess dye using a desalting column pre-equilibrated in Dialysis Buffer.