產(chǎn)品詳情

  • 產(chǎn)品名稱：Spincolumns

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  • 產(chǎn)品廠商：ChromoTek
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Spincolumns
詳情介紹：
Purpose These spin columns can be used to wash and elute your proteins.
Characteristics These spin columns can be used to wash and elute your proteins. Instead of removing or collecting the supernatant you can just use spin columns. Either discard the flow through during the washing steps or keep the flow-through after adding the elution buffer. The spin columns are making the handling easier.
Comment

Spin columns can be used in addition to agarose coupled Nano-Traps.
Sample Collection Harvest cells:
For one immunoprecipitation reaction the use of 10^6 - 10^7 mammalian cells (approx. one 10 cm dish) is recommended. To harvest adherent cells, aspirate growth medium, add 1 ml ice-cold PBS to cells and scrape cells from dish. Transfer cells to a pre-cooled tube, spin at 500 g for 3 min at +4°C and discard supernatant. Wash cell pellet twice with ice-cold PBS, gently resuspending the cells.

Lyse cells
  • 1. Resuspend cell pellet in 200 μL ice-cold lysis buffer by pipetting or using a syringe.
    note: Supplement lysis buffer with protease inhibitors and 1 mM PMSF (not included). optional for nuclear/chromatin proteins: Use RIPA buffer supplemented with 1 mg/mL DNase, 2.5 mM MgCl2, protease inhibitors and 1 mM PMSF (not included).
  • 2. Place the tube on ice for 30 min with extensively pipetting every 10 min.
  • 3. Centrifuge cell lysate at 20.000x g for 10 min at +4°C. Transfer lysate to a pre-cooled tube. Add 300 μl dilution buffer to lysate. Discard pellet.
    note: At this point cell lysate may be put at -80°C for long-term storage. optional: Add 1 mM PMSF and protease inhibitors (not included) to dilution buffer

We recommend that during incubation with the beads the final concentration of detergents does not exceed 0.2% to avoid unspecific binding to the matrix. If required, use more dilution buffer to dilute the supernatant accordingly.
Assay Procedure

Equilibrate beads:

  • 4. Remove the upper cap of a new spin column and snap of the plug from the bottom of the spin column (Keep cap and plug!). Place the spin column in a 2 ml tube. Vortex Nano-Trap?_A beads and pipette 25 μL bead slurry into the spin column. Immediately add 500 μl ice-cold dilution buffer. Centrifuge at 100x g for 5-10 sec. Discard flow-through and repeat wash twice. Close column with the bottom plug.

Bind protein:
  • 5. Add diluted lysate (step 3) to equilibrated Nano-Trap?_A beads (step 4). If required, save 50 μL of diluted lysate for immunoblot analysis. Secure the top of the spin column. Tumble end-over-end for 1 hour at 4°C.
  • 6. Remove the bottom cap from the spin column and place it in a new 2 ml tube. Centrifuge at 100x g for 5-10 sec. If required, save 50 μL flow-through for immunoblot analysis. Discard remaining flow-through.

Wash beads:
  • 7. Resuspend Nano-Trap?_A beads in 500 μL ice-cold dilution buffer. Place spin column in a 2 mL tube and centrifuge at 100x g for 5-10 sec. Discard flow-through and repeat wash twice.Close column with the bottom plug.
    optional: Increase salt concentration in the second washing step up to 500 mM.

Elute proteins:
  • 8. Add 50 μl elution buffer (0.2 M glycine, pH 2.5) to Nano-Trap?_A beads. Pipette Nano-Trap?_A beads up and down for 30 sec. Remove bottom plug of the spin column and place it in a new 2 mL tube containing 5 μL 1M Tris base pH 10.4. Centrifuge at 1000x g for 30-60 sec. To increase elution efficiency this step can be repeated.
Restrictions For Research Use only
Storage RT