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Conjugation of Alkyne- or Azide-modified Proteins with Dye Azides or Alkynes

Intended for the CuAAC reaction with proteins, protein labeling buffer protects biomolecules from being damaged by reactive oxygen species. This reaction can be used for proteins with alkyne or azide groups, as well as cells or components of cell lysates metabolically labeled with azide groups.

Azide is conjugated to terminal alkyne, resulting in a five-membered heterocycle (1,2,3-triazole). Both groups (azide and alkyne) are extremely rarely found in natural biomolecules, so the reaction is highly specific and effective to handle various tasks.

Scheme of copper catalyzed Click chemistry alkyne azide cycloaddition

The reaction proceeds in the presence of copper (I) compounds and almost does not depend on pH. Optimized for operations with proteins, protein labeling buffer contains a salt of copper (II) (a stable precursor of catalytically-active copper (I) compounds), triethylammonium acetate pH 6.8, water-soluble THPTA ligand, and aminoguanidine. It is recommended to use a freshly prepared solution of ascorbic acid to reduce copper (II). THPTA ligand in protein labeling buffer speeds up the reaction by stabilizing catalytically-active compounds of copper (I). The presence of THPTA ligand also allows protein labeling in water medium (without organic solvents) and, owing to stabilization of copper (I) oxidation degree, minimizes production of reactive oxygen species (RAS) and prevents them from damaging proteins by oxidizing histidine, methionine and cysteine. Aminoguanidine in protein labeling buffer prevents chemically reactive aldehydes (dehydroascorbate hydrolysis products) from binding to side chains of arginine, N-terminal cysteine, and lysine.

For this reaction, you will need alkyne- or azide-modified protein in azide sodium-free buffer, dye azide or alkyne, 1.5х protein labeling buffer, and ascorbic acid. It is recommended to perform steps 6 to 9 under an inert gas (nitrogen or argon).

Protocol

We recommend the following protocol for conjugation of modified proteins with dye derivatives:

Determine total reaction volume based on the amount of modified protein to be used:

! The volume of alkyne- or azide-modified protein solution should be not more than 1/3 of total reaction volume.

Total reaction volume, µL	Amount of protein
100	4 to 20 nmol
200	20 to 40 nmol
400	40 to 80 nmol
600	80 to 600 nmol

Calculate volumes of the reagents for the labeling reaction using the table below:

Reagent	Volume, µL	Concentration of stock solution
Dye azide or alkyne	(amount of protein [nmol]) × 0.3*	10 mM in DMSO or water
Protein labeling buffer	(total reaction volume [µL]) × 0.67	1.5х
Activator (ascorbic acid)	(total reaction volume [µL]) × 0.02	50 mM in water
Water	(total reaction volume [µL] – volume of dye solution [µL] – volume of protein labeling buffer [µL] – volume of activator solution [µL])	—

* the dye excess may vary depending on the number of azide or alkyne groups on the protein molecule. Calculations in the table are shown for 3x excess of the dye. For 1.5–10x excess of the dye, multiply protein amount (nmol) by 0.15–1. But remember that if you use an azide or alkyne that is not soluble in water, in a big excess it may precipitate from the reaction mixture. Use water-soluble dyes, for example, sulfonated cyanine dyes sulfo-Cyanine.

Prepare stock solution of dye azide or alkyne (10 mM in DMSO or water, for water-soluble alkynes and azides) and activator (ascorbic acid, 50 mM in water).
Bear in mind that ascorbic acid is readily oxidizable in air. Use only a freshly prepared solution of activator (the solution is stable within 1 day). To prepare stock solution, dissolve 10 mg of ascorbic acid in 1.1 mL of water.
Add protein labeling buffer to modified protein solution and vortex.
Add the calculated volume of stock solution of dye azide or alkyne and vortex well again.
(recommended) Degas the mixture to remove oxygen. To do so, connect a disposable pipette tip to a plastic or silicone tubing connected to the pressure regulator of a gas cylinder with inert gas (argon or nitrogen). Turn on a very weak gas flow and put the tip down in the tube so that it can be 3–10 mm higher than the liquid level avoiding touching the liquid and tube walls. The gas flow should make a swirl in the liquid without spattering it. Keep the tip in this position for 10–20 seconds.
If several labeling reactions are run simultaneously, a centrifugal concentrator can be used for degassing. To do so, place the tubes in the concentrator, turn on rotation, turn on vacuum for 30–40 seconds, then turn off vacuum while feeding inert gas to the input of the system.
Add activator solution (ascorbic acid), then purge the tube with inert gas for a few seconds and close it.
Vortex the solution.
Allow the mixture to stand at room temperature for 8–16 h.
Use dialysis or size-exclusion chromatography to isolate the dye-protein conjugate.

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12630	1 mg	$105	现货
22630	5 mg	$165	现货
42630	25 mg	$285	现货
52630	50 mg	$495	现货
62630	100 mg	$880	现货

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11830	1 mg	$125	现货
21830	5 mg	$325	现货
41830	25 mg	$950	现货
51830	50 mg	$1625	现货
61830	100 mg	$2090	现货

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S7830	250 ug	–	现货
A7830	1 mg	$140	现货
B7830	5 mg	$450	现货
D7830	25 mg	$1460	现货
E7830	50 mg	$1890	现货
F7830	100 mg	$2640	现货

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1C930	1 mg	$165	现货
2C930	5 mg	$640	现货
4C930	25 mg	$1640	现货
5C930	50 mg	$2090	现货
6C930	100 mg	$2840	现货

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1A930	1 mg	$165	现货
2A930	5 mg	$640	现货
4A930	25 mg	$1640	现货
5A930	50 mg	$2090	现货
6A930	100 mg	$2840	现货

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1B930	1 mg	$165	现货
2B930	5 mg	$640	现货
4B930	25 mg	$1640	现货
5B930	50 mg	$2090	现货
6B930	100 mg	$2840	现货

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AG430	1 mg	$140	现货
BG430	5 mg	$450	现货
DG430	25 mg	$1460	现货
EG430	50 mg	$1890	现货
FG430	100 mg	$2640	现货

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15430	1 mg	$125	现货
25430	5 mg	$260	现货
35430	10 mg	$325	现货
45430	25 mg	$510	现货
55430	50 mg	$895	现货

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A54B0	1 mg	$125	现货
B54B0	5 mg	$260	现货
C54B0	10 mg	$325	现货
D54B0	25 mg	$510	现货
E54B0	50 mg	$895	现货
F54B0	100 mg	$1490	现货

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C4130	10 mg	$139	现货
D4130	25 mg	$285	现货
E4130	50 mg	$445	现货
F4130	100 mg	$765	现货

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35130	500 uL, 10 mM/DMSO	$97	现货
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A5130	1 mg	$69	现货
B5130	5 mg	$97	现货
C5130	10 mg	$139	现货
D5130	25 mg	$285	现货
E5130	50 mg	$445	现货
F5130	100 mg	$765	现货

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15230	100 uL, 10 mM/DMSO	$160	现货
35230	500 uL, 10 mM/DMSO	$420	现货
45230	1 mL, 10 mM/DMSO	$595	现货
A5230	1 mg	$115	现货
D5230	25 mg	$510	现货
E5230	50 mg	$895	现货
F5230	100 mg	请查询	现货

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11430	1 mg	$125	现货
21430	5 mg	$260	现货
31430	10 mg	$325	现货
41430	25 mg	$510	现货
51430	50 mg	$895	现货
61430	100 mg	$1490	现货

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A14B0	1 mg	$125	现货
B14B0	5 mg	$260	现货
C14B0	10 mg	$325	现货
D14B0	25 mg	$510	现货
E14B0	50 mg	$895	现货
F14B0	100 mg	$1490	现货

目录号	数量	价格	提前通知时间
12430	1 mg	$125	现货
22430	5 mg	$260	现货
32430	10 mg	$325	现货
42430	25 mg	$410	现货
52430	50 mg	$895	现货
62430	100 mg	$1490	现货

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A24B0	1 mg	$125	现货
B24B0	5 mg	$260	现货
C24B0	10 mg	$325	现货
D24B0	25 mg	$510	现货
E24B0	50 mg	$895	5 天
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32030	500 uL, 10 mM/DMSO	$199	现货
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A2030	1 mg	–	现货
B2030	5 mg	$199	现货
C2030	10 mg	$297	现货
D2030	25 mg	$495	现货
E2030	50 mg	$895	现货
F2030	100 mg	$1490	现货

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11030	100 uL, 10 mM/DMSO	–	20 天
31030	500 uL, 10 mM/DMSO	$199	现货
41030	1 mL, 10 mM/DMSO	$495	现货
A1030	1 mg	–	现货
B1030	5 mg	$199	现货
C1030	10 mg	$297	现货
D1030	25 mg	$495	现货
E1030	50 mg	$895	现货
F1030	100 mg	$1490	现货

目录号	数量	价格	提前通知时间
A10B0	1 mg	$125	现货
B10B0	5 mg	$260	现货
C10B0	10 mg	$325	现货
D10B0	25 mg	$510	现货
E10B0	50 mg	$895	现货
F10B0	100 mg	$1490	现货

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14030	100 uL, 10 mM/DMSO	–	现货
34030	500 uL, 10 mM/DMSO	$199	现货
44030	1 mL, 10 mM/DMSO	$495	现货
A4030	1 mg	–	现货
B4030	5 mg	$199	现货
C4030	10 mg	$297	现货
D4030	25 mg	$495	现货
E4030	50 mg	$895	现货
F4030	100 mg	$1490	现货

目录号	数量	价格	提前通知时间
A70B0	1 mg	$125	现货
B70B0	5 mg	$260	现货
C70B0	10 mg	$325	现货
D70B0	25 mg	$510	现货
E70B0	50 mg	$895	现货
F70B0	100 mg	$1490	现货

目录号	数量	价格	提前通知时间
13030	100 uL, 10 mM/DMSO	–	现货
33030	500 uL, 10 mM/DMSO	$199	现货
43030	1 mL, 10 mM/DMSO	$495	现货
A3030	1 mg	–	现货
B3030	5 mg	$199	现货
C3030	10 mg	$297	现货
D3030	25 mg	$495	现货
E3030	50 mg	$895	现货
F3030	100 mg	$1490	现货

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Conjugation of Alkyne- or Azide-modified Proteins with Dye Azides or Alkynes

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A30B0	1 mg	$125	现货
B30B0	5 mg	$260	现货
C30B0	10 mg	$325	现货
D30B0	25 mg	$510	现货
E30B0	50 mg	$895	现货
F30B0	100 mg	$1490	现货

目录号	数量	价格	提前通知时间
16030	100 uL, 10 mM/DMSO	–	现货
36030	500 uL, 10 mM/DMSO	$199	现货
46030	1 mL, 10 mM/DMSO	$495	现货
A6030	1 mg	–	现货
B6030	5 mg	$199	现货
C6030	10 mg	$297	现货
D6030	25 mg	$495	现货
E6030	50 mg	$895	现货
F6030	100 mg	$1490	现货

目录号	数量	价格	提前通知时间
A60B0	1 mg	$125	现货
B60B0	5 mg	$260	现货
C60B0	10 mg	$325	现货
D60B0	25 mg	$510	现货
E60B0	50 mg	$895	现货
F60B0	100 mg	$1490	现货

目录号	数量	价格	提前通知时间
15030	100 uL, 10 mM/DMSO	–	现货
35030	500 uL, 10 mM/DMSO	$199	现货
45030	1 mL, 10 mM/DMSO	$495	现货
A5030	1 mg	–	现货
B5030	5 mg	$199	现货
C5030	10 mg	$297	现货
D5030	25 mg	$495	现货
E5030	50 mg	$895	现货

目录号	数量	价格	提前通知时间
A50B0	1 mg	$125	现货
B50B0	5 mg	$260	现货
C50B0	10 mg	$325	现货
D50B0	25 mg	$510	现货
E50B0	50 mg	$895	现货
F50B0	100 mg	$1490	现货

目录号	数量	价格	提前通知时间
A41B0	1 mg	–	现货
B41B0	5 mg	$100	现货
C41B0	10 mg	$150	现货
D41B0	25 mg	$295	现货
E41B0	50 mg	$450	现货
F41B0	100 mg	$780	现货

目录号	数量	价格	提前通知时间
A51B0	1 mg	–	现货
B51B0	5 mg	$100	现货
C51B0	10 mg	$150	现货
D51B0	25 mg	$295	现货
E51B0	50 mg	$450	现货
F51B0	100 mg	$780	现货

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A1130	1 mg	$110	现货
B1130	5 mg	$210	现货
C1130	10 mg	$310	现货
D1130	25 mg	$410	现货
E1130	50 mg	$695	现货
F1130	100 mg	$1190	现货

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A4430	1 mg	$110	现货
B4430	5 mg	$210	现货
C4430	10 mg	$310	现货
D4430	25 mg	$410	现货
E4430	50 mg	$695	现货

目录号	数量	价格	提前通知时间
A1230	1 mg	$110	现货
B1230	5 mg	$165	现货
C1230	10 mg	$210	现货
D1230	25 mg	$299	现货
E1230	50 mg	$495	现货