Immunoprecipitation of Fusion Proteins from Cell Extracts us

NanoTag's Selector resins are based on high-affinity single-domain antibody (sdAb) fragments covalently immobilized on 4% cross-linked agarose beads. The innovative, oriented and selective attachment via flexible linkers guarantees optimal accessibility of the sdAbs and in addition largely eliminates batch-to-batch variations. Due to the single-chain nature of sdAbs and their stable and covalent attachment, no leakage of light and heavy chains is observed during elution with SDS sample buffer. Selector resins thus feature high affinity and superior capacity for fusion proteins while showing negligible non-specific background. Selector resins are compatible not only with physiological buffers but also with high stringency buffers (see product-specific compatibility charts) and reducing agents. Selector resins thus provide great freedom to adjust the binding and washing conditions to the experimental needs.

Products

• N0110: MBP Selector,    2000 µl 50% slurry (1000 µl resin)
• N0210: GST Selector,    2000 µl 50% slurry (1000 µl resin)
• N0310: GFP Selector,    2000 µl 50% slurry (1000 µl resin)
• N0410: RFP Selector,    2000 µl 50% slurry (1000 µl resin)
• N0510: TagFP Selector, 2000 µl 50% slurry (1000 µl resin)

Solutions needed

• Lysis buffer: Selector resins are compatible with most common lysis buffers (e.g. RIPA). Please refer to the product-specific compatibility chart on the respective data sheet.
• Washing buffer: Selector resins are compatible with most common washing buffers. Please refer to the product-specific compatibility chart on the respective data sheet.
• TBS: Tris-buffered saline pH 7.4
• 2x SDS sample buffer

Remarks: Selector resins are compatible with most common lysis/washing buffers (e.g. RIPA). For custom buffers please refer to the product-specific compatibility chart.

Protocol using Mini-Spin columns

We recommend using Mini-Spin columns for immunoprecipitation experiments. An alternative batch protocol is found on the following page.

1. Prepare native cell lysates (0.2 to 1.5 ml volume) according to established protocols. For mammalian cells, we recommend using 106 - 108 cells per experiment.
2. Clarify lysate by centrifugation for 10 min at > 14000 x g and 4°C. Take sample for further analysis (input fraction).
3. Equilibrate Selector resin by transferring 20 μl resuspended slurry (10 μl packed beads) into a clean 1.5 ml reaction tube.
4. Add 1 ml lysis buffer and centrifuge for 1 min at 1000 x g and carefully remove supernatant.
5. Repeat step 4 once.
6. Add clarified lysate from step 2 to equilibrated Selector resin obtained in steps 3 - 5.
7. Incubate 1 h at 4°C with head-over-tail rotation.
8. Sediment beads by centrifugation for 1 min at 1000 x g and 4°C. Take sample from supernatant for further analysis (non-bound fraction).
9. Carefully remove supernatant.
10. Resuspend beads in 1 ml lysis buffer and centrifuge for 1 min at 1000 x g.
11. Remove supernatant.
12. Remove bottom plug from Mini-Spin column and place column in 2 ml reaction tube.
13. Resuspend beads in 200 μl lysis buffer, transfer suspension to Mini-Spin column.
14. Wash out beads sticking to tube with 200 μl lysis buffer and transfer to column.
15. Centrifuge column for 1 min at 1000 x g, discard flow-through.
16. Wash twice with 400 μl washing buffer and centrifuge for 1 min at 1000 x g.
17. Wash once with 400 μl TBS and centrifuge for 1 min at 3000 x g.
18. Attach bottom plug and place Mini-Spin column in a clean 1.5 ml reaction tube.
19. Resuspend Selector resin in 50 μl 2 x SDS sample buffer.
20. Heat Mini-Spin column to 95°C for 2 min.
21. Remove bottom plug and centrifuge for 1 min at 3000 x g.
22. Boil collected eluate for 5 min at 95°C and analyze by SDS-PAGE.

Batch protocol

Due to the more effective washing steps, we recommend using Mini-Spin columns. However, in some cases a batch protocol may be more applicable. 

1. Prepare native cell lysates (0.2 to 1.5 ml volume) according to established protocols. For mammalian cells, we recommend using 106 - 108 cells per experiment.
2. Clarify lysate by centrifugation for 10 min at > 14000 x g and 4°C. Take sample for further analysis (Input fraction).
3. Equilibrate Selector resin by transferring 20 μl resuspended slurry (10 μl packed beads) into a clean 1.5 ml reaction tube.
4. Add 1 ml lysis buffer and centrifuge for 1 min at 1000 x g and carefully remove supernatant.
5. Repeat step 4 once.
6. Add clarified lysate from step 2 to equilibrated Selector resin obtained in step 3.
7. Incubate 1 h at 4°C with head-over-tail rotation.
8. Sediment beads by centrifugation for 1 min at 1000 x g and 4°C. Take sample from supernatant for further analysis (non-bound fraction).
9. Carefully remove supernatant.
10. Resuspend beads in 1 ml lysis buffer and centrifuge for 1 min at 1000 x g.
11. Wash beads 2 - 3 times with washing buffer.
12. Wash beads once with TBS.
13. Transfer beads in clean 1.5 ml reaction tube and centrifuge for 1 min at 3000 x g.
14. Carefully and completely remove supernatant.
15. Resuspend Selector resin in 50 μl 2 x SDS sample buffer and heat for 5 min to 95°C.
16. Centrifuge for 1 min at 3000 x g.
17. Collect supernatant and analyze by SDS-PAGE.