SureCapture-pY Enrichment Quick-Start Manual

Product Description and Intended Use

Thank you for purchasing the Axoiya Inc. AxoBind SureCapture-pY enrichment kit. SureCapture-pY is a magnetically coupled high-affinity phosphotyrosine (pY) SuperBinder which offers superior and specific enrichment of pY peptides over that of standard pY enrichment methods such as immobilized metal-affinity chromatography (IMAC) or pY antibodies. This product is for research purposes only and not for diagnostic use. 

NOTES (Upon arrival, store the kit at 4 °C, shelf life = 4 months)

  • Use of low-binding pipette tips is recommended for handling the SureCapture-pY beads
  • The protocol below is designed for enriching peptides from 200-1000 μg tryptic digests
  • Use of KingFisher 96 or similar is recommended for handling of Steps 4 and 5 
  • Cleanup with an SPE AxoBind-tip (included) can be performed in microcentrifuge with Tip Adapter, up to 700xg for 1 min or until all solution has passed through 
  • For more details, such as KingFisher 96 recommended settings, proceed to the website www.axoiya.com 

 

PROTOCOL

    1. Re-suspend protein lysate tryptic digest
        a. Add 205 μL Resuspension Buffer to your dried clean tryptic digest
        b. Vortex to ensure dissolution
    2. Prepare AxoBind Enhancer
        a. Remove AxoBind Enhancer from 4°C storage
    3. Prepare Peptides
        a. Add 5 μL AxoBind Enhancer to resuspended peptides
            i.  If enriching more than 200 μg peptides, scale up volume of AxoBind Enhancer accordingly
            ii.  Store leftover AxoBind Enhancer at 4 °C for future experiments
        b. Incubate 15 min, RT, 900 RPM
        c. Centrifuge 10 min at ≥15,000 x g
    4. Prepare SureCapture-pY
        a. Remove SureCapture-pY from 4 °C Storage
        b. Add 100 μL SureCapture-pY to 500 μL AxoBind Buffer 1: mix 15 sec, resolve (using a magnet to clarify beads from the supernatant) and transfer beads (dispose of supernatant if performing manually) to next step
            i.  Store remaining SureCapture-pY at 4 °C
        c. Wash 3 x 600 μL AxoBind Buffer 1: mix 2 min, resolve and transfer to next step
    5. Enrich
        a. Transfer 200 μL clarified peptides to SureCapture-pY beads: mix 1 hr, resolve and transfer to next step
        b. Wash 3 x 600 μL AxoBind Buffer 1: mix 2 min, resolve and transfer to next step
        c. Wash 1 x 600 μL AxoBind Buffer 2: mix 2 min, resolve and transfer to next step
        d. Elute peptides with 200 μL Elution Buffer: mix 15 min, resolve and use supernatant for Step 6 
    6. Cleanup
        a. Condition SPE AxoBind-tip with 100 μL 100% methanol
        b. Condition SPE AxoBind-tip with 100 μL SPE Conditioning Buffer
        c. Equilibrate SPE AxoBind-tip with 100 μL SPE Wash Buffer
        d. Apply enriched SureCapture-pY peptide sample
        e. Wash SPE AxoBind-tip with 100 μL SPE Wash Buffer
        f. Transfer AxoBind-tip+adapter to fresh collection tube
        g. Elute AxoBind-tip with 2 x 70 μL SPE Elution Buffer
    7. SpeedVac until dry
        a. Dried sample can be stored in freezer until ready for nano-LC-MS/MS analysis
    8. Resuspend and analyze by nano-LC-MS/MS 
        a. Resuspend enriched peptides in 12 μL 0.2% FA/2% ACN/H2O and transfer to injection vials for nano-LC-MS/MS. Inject 10 μL and run on a 120-minute gradient of 1-35% ACN/0.1% FA. 


For technical support, please contact Axoiya helpdesk at email support@axoiya.com