Azido-PEG3-Maleimide kit

Azido-PEG3-Maleimide degrades quickly (hours) at room temperature. This product is provided as a
two-component kit. The stock solution of Azido-PEG3-Maleimide is prepared with a
straightforward mix-and-stir protocol in 30 min.

- Molecules to be reacted with Azido-PEG3-Maleimide must have free (reduced) sulfhydryls. - Do not use buffers that contain sulfhydryl-containing components (e.g., DTT), and azides
- The maleimide group reacts predominantly with free sulfhydryls at pH 6.5-7, forming stable
thioether bonds. At pH values > 7.0, reactivity toward primary amines and hydrolysis of the
maleimide groups can occur. At pH 7, the maleimide group is ~1,000 times more reactive
toward a free sulfhydryl than to an amine.


Additional Materials Required

- Water miscible organic solvent such as dimethyl sulfoxide (DMSO) or dimethyl formamide
(DMF)
- Reducing reagents such as TCEPT or Immobilized TCEP Disulfide Reducing Gel
- Reaction buffer: Phosphate.buffered saline (PBS) or other sulfhydryl.free buffer at pH 6.5.7.0.
Include 5.10 mM EDTA to help prevent the re.oxidation of disulfides by trace divalent metals.
- (Optional): Quenching buffer: concentrated (0.5.1 M) cycteine, DDT or other thiol containing
reducing agents
- Spin Desalting Columns


Preparation of Azido-PEG3-Maleimide Stock Solution

1. Add dry water.miscible organic solvent to Azido-PEG3-amine (vial #2) and shake for ca. 30 seconds.


2. While keeping the Maleimide-NHS ester (vial #1, white solid) under a dry atmosphere (e.g. with
nitrogen) slowly add a solution of Azido-PEG3-Amine (vial #2) with stirring or shaking, and then stir
or shake for 30 minutes at room temperature. The progress of the reaction can be followed by TLC.

3. Stock solution of Azido-PEG3-Maleimide is ready to use. At this stage the product is stable if stored
at -20C or lower for short periods of time (hours).

4. The concentration of azide-PEG3-maleimide stock solution is ca. 120 mM (provided that 2.5mL of
water-miscible organic solvent is added to Azido-PEG3-Amine (vial #2)). The reaction produces ca.
0.3 mmol of Azido-PEG3-Maleimide

5. TLC data: The solvent is typically something like methanol: methylene chloride 1:20 or 4 ml: 10
drops, run on a silica gel normal phase plate and developed with a potassium permanganate spray.
E.g. the Rf of the Azido-PEG3-Maleimide is slightly lower one of the Maleimide-NHS ester. When the
reaction is complete, it will be one clean spot on the plate.


Procedure for Labeling Proteins

1. If required, buffer exchange the protein sample into phosphate reaction buffer at 1.5 mg/mL using a
spin desalting column.

2. Add stock solution of TCEP to the protein solution at final concentration of 20 mM, pipette up and
down several times to mix.

3. Incubate the reaction to for 30 minutes.

4. Buffer exchange TCEP reduced protein into reaction buffer. If a reaction buffer does not contain
EDTA, add immediately stock solution of EDTA to a solution of reduced protein at final concentration
of 5.10 mM.

5. Add a 20-fold molar excess of freshly prepared maleimide reagent to the protein sample.

6. Incubate reaction mixture for 1.4 hour at room temperature or for 2.8 hours at 4°C.

Note: Many proteins will precipitate when the DMF or DMSO concentration exceeds 10% of the final
reaction volume; if protein solubility is not an issue, there is no limit to the DMF or DMSO
concentration that may be used.

7. Remove the excess reagent by desalting the labeled protein through a spin desalting column or by
dialysis.


Procedure for click reaction

In order to perform the Cu(I)-catalyzed azide-alkyne click reaction (CuAAC) at least one
additional component is required – copper(I) catalyst. However, the general thermodynamic instability
of Cu(I), which results in its easy oxidation to inactive Cu (II) usually requires the copper catalyst to be
prepared with an appropriate chelating ligand.

The selection of partial protocol for CuAAC reaction depends mainly on two factors, including
nature of alkyne-modified biopolymer (e.g. cell lysis, fixed cells) and availability of chelating ligand.

Click Chemistry Tools offers reaction kits that enable researchers to perform Cu(I).catalyzed
azidealkyne click reaction. The kits contain detailed procedure and all reagents required to perform up
to 25 CuAAC reactions.