» Sign in / Register

Click Chemistry Capture Kit

Cat. No. Amount Price (EUR) Buy / Note
CLK-1065 1 kit 393,09 Add to Basket/Quote Add to Notepad

For research use only!

Shipping: shipped at ambient temperature

Storage Conditions: store components as indicated on data sheet

Shelf Life: 12 months after date of delivery

Description:
The Click Chemistry Capture Kit provides all necessary reagents to covalently capture specific sub-classes of proteins by a Cu(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC). The proteins of interest need to be metabolically, enzymatically or chemically azido- or alkyne-tagged. Subsequently, the resin containing the covalently attached proteins can be washed with high stringency, virtually eliminating any non-specifically bound proteins. Upon protease digestion, this yields a highly pure peptide pool that is ideal for mass spectroscopy (e.g. LC MS/MS) based analysis.

Content:

  • 7 ml Lysis buffer
    - store at 4 °C
  • 4.8 g Urea
    - store at ambient temperature
  • 1.5 ml Additive 1
    - store at 4 °C
  • 0.5 ml Copper (II) Sulfate (100 mM)
    - store at ambient temperature
  • 400 mg Additive 2
    - store at ambient temperature
  • 200 ml Agarose wash buffer
    - store at 4 °C
  • 10 Empty spin columns
    - store at ambient temperature

Materials required but not provided for capturing of Azide-tagged proteins

  • 5-20 mg azido- or alkyne-tagged cell or tissue extract
  • Alkyne or Azide agarose resin
  • Unlabeled negative control cells or tissue
  • Sample rotator
  • Table top centrifuge
  • Protease Inhibitor
  • 2 ml microcentrifuge tubes
  • aqua bidest.
  • Probe sonicator

Materials required but not provided for on-resin digestion with protease

  • DTT
  • Iodoacetamide
  • 8 M Urea, 100 mM Tris, pH 8
  • Acetonitrile
  • Mass-spec grade trypsin
  • 0.1 % TFA
  • C-18 desalting cartridges
  • Digestion Buffer (100 mM Tris, 2 mM CaCl2, 10 % acetonitrile)
  • Heat block
  • Vacuum concentrator


Preparation of stock solutions

Lysis Buffer (200 mM Tris, 4 % CHAPS, 1 M NaCl,
8M Urea, pH 8.0)

  • Add the solid urea (4.8 g) to the Lysis Buffer (7 ml) provided.
  • Mix the solution on a rotator at room temperature until the urea is completely dissolved (1-2 hours). Store at -4 °C (for up to 1 week) or at -20 °C for 1 year to avoid decomposition of urea.
  • Note: 30 min before starting the enrichment protocol, add 20 μl Protease Inhibitor Cocktail (e.g. Sigma 8340) per ml of Lysis Buffer (sufficient for 50-200 million cells or 5-20 mg tissue extract).

Additive 2

  • Add 2 ml of aqua bidest. to Additive 2 and vortex until fully dissolved.
  • After use, store remaining stock solution at -20°C for up to 1 year.

Protein Enrichment Protocol (per enrichment)

Step 1: Preparation of Alkyne- or Azide-Agarose Resin

  • Mix the 50 % resin slurry until the resin is completely resuspended.
  • Before the resin settles, transfer 200 μl of well-mixed resin with a 1 ml pipette into a clean 2 ml microfuge tube.
  • Add 1.3 ml aqua bidest. to the resin.
  • Pellet the resin by centrifugation for 2 min at 1000 x g.
  • Carefully discard the supernatant leaving approximately 200 μl of settled resin at the bottom of the tube. Take care not to aspirate settled resin.

Step 2: Lysate Preparation

  • Add 1 ml Lysis Buffer including Protease Inhibitor (see Preparation of stock solutions) to each azide- or alkyne-containing cell or tissue extract (5-20 mg protein) in a 2 ml microfuge tube.
  • Incubate the lysis mixture on ice for 5-10 min.
  • While on ice, sonicate the mixture using a probe sonicator by applying two 3 second pulses. Take care not to overheat the sample.
  • Repeat the sonication on ice until the lysate is no longer viscous (e.g. viscosity of water).
  • Centrifuge the lysate at 10,000 x g for 5 min.
  • Place the lysate back on ice until performing the click reaction.

Step 3: Preparation of 2x Copper Catalyst Solution

  • Prepare 1 ml of 2x Copper Catalyst Solution per enrichment reaction as follows:
    - 860 μl Aqua bidest.
    - 100 μl Additive 1
    - 20 μl Copper (II) Sulfate Solution
    - 20 μl Additive 2
  • Voretex the solution

Step 4: Lysate/Agarose Click Reaction

  • Assemble the click reaction in a 2 ml microfuge tube as follows:
    - 200 μl washed Alkyne or Azide Agarose resin (from step 1)
    - 800 μl cell or tissue lysate (from step 2)
    - 1000 μl 2x Copper Catalyst Solution (from step 3)
  • Rotate end-over end on sample rotator for 16-20 hours

Step 5: Reduction & Alkylation of Resin Bound Proteins

  • Warm the Agarose wash buffer to room temperature before starting. Ensure that the solution is homogenous and clear before use.
  • Centrifuge the agarose resin for 1 min at 1000 x g. Carefully discard the supernatant.
  • Add 1.8 ml of aqua bidest. to the resin, centrifuge at 1000 x g and carefully discard the supernatant. This water wash step prevents clumping of the resin caused by interaction of residual Lysis Buffer with the SDS in Agarose wash buffer.
  • Add 1 ml Agarose wash buffer and 10 μl of 1 M DTT to the resin. Vortex briefly to resuspend the resin.
  • Heat the resin at 70 °C for 15 min on a heat block, then cool to room temperature for 15-30 min.
  • Centrifuge resin for 5 min at 1000 x g and carefully discard the supernatant.
  • Prepare 1 ml of a 40 mM iodoacetamide solution per enrichment reaction by dissolving 7.4 mg of iodoacetamide in 1 ml of Agarose wash buffer.
  • Add 1 ml 40 mM iodoacetamide solution to the resin, vortex to resuspend the resin and incubate the reaction in the dark for 30 min at room temperature.


Step 6: Resin Wash

  • Agarose wash buffer is used for stringent removal of non-specifically bound proteins. It is critical to remove residual SDS by exhaustive washing with 8 M urea and 20 % acetonitrile prior to mass spectrometry analysis.
  • Twist off the spin column's bottom closure and remove the cap.
  • Use a 1 ml pipette to resuspend the resin, then transfer the resin to a spin column.
  • Rinse the resin tube with 0.5 ml H2O and also transfer this volume to the same spin column.
  • Add 2 ml of Agarose wash buffer to the spin column and centrifuge at 1000 x g for 1 min. Repeat this step 5 times.
  • Add 2 ml of 8 M Urea/100 mM Tris, pH 8 to the spin column and centrifuge at 1000 x g for 1 min. Repeat this step 5-10 times.
  • Add 2 ml of 20 % acetonitrile to the spin column and centrifuge at 1000 x g for 1 min. Repeat this step 5-10 times.

Protease Digestion of Resin-Bound Proteins

  • Cap the bottom of the spin column and add 500 μl of Digestion Buffer to the resin.
  • Use a 1 ml pipette to resuspend the resin in the spin column and transfer the resin to a clean tube.
  • Rinse the spin column with 0.5 ml additional Digestion Buffer and add this rinse to the already transferred resin.
  • Pellet the resin by centrifugation for 5 min at 1000 x g. Discard the supernatant, but leave approximately 200 μl of Digestion Buffer in the tube above the resin. Take care not to aspirate the resin.
  • Add 10 μl of 0.1 μg/μl trypsin to the resin slurry and mix gently. Incubate at 37 °C for 6 hours to overnight.

Preparation of Digest for Mass Spectrometry Analysis

  • Pellet the resin by centrifugation for 5 min at 1000 x g and carefully transfer the digested supernatant to a clean tube.
  • Add 500 μl aqua bidest. to the resin. Vortex briefly and pellet the resin by centrifugation for 5 min at 1000 x g.
  • Transfer the supernatant to the already transferred digest supernatant.
  • Add additional aqua bidest. to the digest to a final volume of 1 ml (Please note: This dilutes the acetonitrile concentration to 2 %).
  • Acidify the diluted digest by adding 2 μl of TFA.
  • Desalt the digest on a C-18 cartridge using vacuum or gravity flow. Allow each solution to completely flow through the cartridge before adding the next solution.
  • Add 1 ml of 50 % acetonitrile/0.1 % TFA to the cartridge and discard the effluent.
  • Add 1 ml of 0.1 % TFA to the cartridge and discard the effluent. Repeat one more time.
  • Add the acidified, diluted digest to the cartridge and discard the effluent.
  • Add 1 ml of 0.1 % TFA to the cartridge and discard the effluent. Repeat one more time.
  • Place a clean 1.5 ml tube below the C-18 cartridge outlet.
  • Elute the peptides into a clean 1.5 ml tube by adding 700 μl of 50 % acetonitrile/0.1 % TFA to the C-18 cartridge.
  • Dry the eluate containing the desalted peptide digest in a vacuum concentrator. Store at -20 °C until MS analysis.


Troubleshooting

Problem: Low yield of enriched proteins

  • Possible reason: Inefficient protein capture or low abundance of azido- or alkyne-tagged proteins
    - Increase the lysate concentration (use more cells) or pre-enrich the proteins (e.g. soluble lysate, membrane lysate, lectin enrichment, etc.).
    - Confirm peptide recovery by measuring A280 after digestion
  • Possible reason: Inefficient digestion of resin-bound proteins
    - Use high quality trypsin

Problem: High background with unlabeled control cells

  • Possible reason: Insufficient washing of resin
    - Increase column washes
    - Use only high purity reagents
    - Prepare fresh filtered buffers
    - Ensure proper preparation of copper catalyst solution

Problem: Signal suppression during MS analysis

  • Possible reason: SDS contamination in the digest
    - Wash the resin thoroughly after the Agarose wash buffer wash with another buffer such as 8 M urea and 20 % acetonitrile to remove all traces of SDS detergent (Step 6)

Product Citations:
Please click the black arrow on the right to expand the citation list. Click publication title for the full text.