Click kit for UV-photoactive probes

For research use only!

Shipping: shipped at ambient temperature
Storage Conditions: store components as indicated on data sheet
Shelf Life: 6 months after the date of delivery

Description:
The Click Chemistry Capture Kit provides all necessary reagents to covalently link specific 3Px probes by a Cu(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC) with the azide-PEG-biotin followed by pull-down of the tagged proteins by magnetic beads.  After separation, proteins can be processed for mass spectroscopy (e.g. LC MS/MS) or immunoblotting analysis.

Content (6 reactions):

  • Lysis buffer – store at 4 °C
  • Additive 1- store at 4 °C
  • Copper (II) Sulfate (100 mM)- store at ambient temperature
  • Additive 2- store at ambient temperature
  • azide-PEG-biotin
  • Buffer W
  • eMagSi magnetic beads

Step 1: Preparation of the lysis buffer

Keep the required optimal volume of lysis buffer on ice and add the following components (not provided): sodium deoxycholate (1% final concentration), 5 U/mL DNase I and 200 U/mL RiboLock RNase Inhibitor.

Step 2 (optional): To block ribosomes on the mRNA and to reduce ribosome dissociation when the 3Px binds, it is suggested to treat the cells with at least 10 μg/mL of cycloheximide for 5 min at 37°C before lysis. We recommend using cells at 70%-80% confluence.

Step 3a: Reaction of the 3Px probe in complete media

  • Treat the cells with the 3Px probe for 10 min at  37°C.
  • Wash with cold PBS
  • Placed on ice and irradiated under a UV lamp (BLX-365, 5 x 8 W) at 365 nm for 5 min (0.75 J/cm2), followed by lysis.

Step 3b: Reaction in the cell lysate

  • Lysis and precipitation of the nuclei and cellular debris by centrifugation at 18000 g and 4°C for 5 min
  • Collect the supernatant and dilute it to A260 = 1 a.u/µL with buffer W (10 mM NaCl, 10 mM MgCl2, 20 µg/mL cycloheximide, and 10 mM Hepes, pH 7 in DEPC water) to a final volume of 150 µL.
  • Incubated with the reactive probe for 1 hour in a 12-well plate
  • UV-irradiate at 365 nm for 5 min (0.75 J/cm2).

Step 4: Washing

After incubation, place the cells on ice and wash them with cold PBS containing CHX 20 mg/mL)
Remove residual PBS with a pipet

Step 5: Lysis

 Perform the lysis by directly adding the lysis buffer to each cell dish. Collect the cell lysate in a 1.5 mL microcentrifuge tube and pellet the nuclei by centrifugation at 20000 g for 5 min. Transfer the supernatant to a new tube and keep it on ice for 20 min. With Nanodrop, check the absorbance of the cell lysate at 260 nm with lysis buffer as blank subtraction

Step 6: Copper-catalyzed “click” reaction in the cell lysates and pull-down assays

Incubate the cell lysate (0.35 mL)  with CuSO4 (2.00 µmol), azide-PEG-biotin (1:1 mole with the 3Px), additive 1 (10 µmol) and additive 2 (100 µmol) overnight a 4°C. For the pull-down experiments, the cell lysates were then incubated with 500 µL of MAGAR-cN (IMMAGINA BioTechnology S.r.l.) for 1 hour at RT.