點(diǎn)擊化學(xué)Protocols
與任何疊氮化物或炔烴點(diǎn)擊反應(yīng)試劑通過(guò)銅催化的點(diǎn)擊反應(yīng)標(biāo)記細(xì)胞裂解液的Protocol
Cell Lysate Labeling 
下載為PDF
This is a general protocol for labeling cell lysate through copper-catalyzed click reaction with any azide or alkyne click detection reagent. This protocol may be used as a starting point for optimization of particular click chemistry procedures. Slight adjustment of azide or alkyne click detection reagent concentrations might be required in case of high background or low signal intensity. Final concentration of an azide or alkyne detection reagent may range from 2 μM to 40 μM. Final concentrations below or above this range are also possible, and should be optimized per the specific application. We recommend starting with a final concentration of 20 μM, and titrating this amount down in case of high background.
1. Prepare the following click solutions:
? 100 mM THPTA ligand in aqueous buffer or water
? 20 mM copper sulfate in water
? 300 mM sodium ascorbate in water
? 1 mM (ca. 1 mg/mL) alkyne or azide labeling reagent in water or DMSO
2. For each azide or alkyne- modified protein lysate sample, add the following to a 1.5 mL microfuge tube, then vortex briefly to mix.
? 50 μL protein lysate (1-5 mg/mL) in protein extraction buffer
? 100 μL PBS buffer
? 4 μL corresponding azide (or alkyne) detection reagent (20 μM final concentration)
3. Add 10 μL of 100 mM THPTA solution, vortex briefly to mix.
4. Add 10 μL of 20 mM CuSO4 solution, vortex briefly to mix.
5. Add 10 μL of 300 mM sodium ascorbate solution to initiate click reaction, vortex briefly to mix.
6. Protect reaction from light and allow click reaction to incubate for 30 minutes at room temperature.
7. Proteins in lysate are now click labeled and ready for downstream processing and/or analysis.
Preparation of Samples for Gel Analysis
1. Add 600 μL methanol to 200 μL reaction mixture, vortex briefly.
2. Add 150 μL chloroform, vortex briefly.
3. Add 400 μL dH2O, vortex briefly.
4. Centrifuge for 5 minutes at 13,000-20,000 x g, carefully remove upper aqueous layer without disturbing interface layer containing proteins. Note-upper aqueous layer may either be colorless or contain color depending on detection reagent.
5. Add 450 μL methanol, vortex briefly.
6. Centrifuge for 5 minutes at 13,000-20,000 x g to pellet protein. Carefully, remove and discard supernatant.
7. Add 450 μL methanol, vortex briefly. Repeat step 6.
8. Open the lid to microfuge tube and allow protein pellet to air-dry for at least 15 minutes.
9. Cap and store labeled sample at -200 C until ready for use.
Labeling of Live Cells
See Hong, V., et al (ref. 5) for detailed protocol and suggestions.
References
1. Rostovtsev, V. V., et al (2002). A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes. Angew. Chem. Int. Ed., 41(14), 2596–2599.
2. Torn?e, C.W., et al (2002). Peptidotriazoles on Solid Phase:? [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3- Dipolar Cycloadditions of Terminal Alkynes to Azides J. Org. Chem., 67(9), 3057–3064.
3. Chan, T.R., et al (2004). Polytriazoles as Copper(I)-Stabilizing Ligands in Catalysis. Org. Lett., 6(17), 2853-2855.
4. (a) Presolski, S. I., et al (2011). Copper-Catalyzed Azide–Alkyne Click Chemistry for Bioconjugation. Current Protocols in Chemical Biology. 3(4), 153–162. (b) Hong, V., et al (2009). Analysis and Optimization of Copper-Catalyzed Azide– Alkyne Cycloaddition for Bioconjugation. Angew. Chem. Int. Ed., 48(52), 9879–9883.
5. Hong, V., et al (2010). Labeling Live Cells by Copper-Catalyzed Alkyne?Azide Click Chemistry. Bioconjugate Chem., 21(10), 1912-1916.
