Protocol for Immunoprecipitation of Fusion Proteins from Cel

The ALFA-tag is a novel, highly versatile epitope tag (core sequence SRLEEELRRRLTE) that can be employed for a wide range of life science applications (see Götzke 2019 (1)). For immunoprecipitation of ALFA-tagged target proteins, NanoTag Biotechnologies offers two types of nanobody-based ALFA Selector resins. ALFA SelectorST (for Super-Tight) was optimized to reach the highest possible binding strength. It features a nanobody binding ALFA-tagged targets with an affinity of around 20 pM. Elution of target proteins from ALFA SelectorST  requires acidic or denaturing conditions. ALFA SelectorPE  (for Peptide Elution) displays a nanobody with lower affinity for ALFA-tagged targets (Kd approx. 11 nM), which was optimized for competitive peptide elution under physiological conditions. Both ALFA Selectors are based on cross-linked 4% agarose beads. The site-directed and chemically stable immobilization of nanobodies allows for a high capacity (> 150 µM target protein, e.g. 4.5 mg GFP-ALFA per ml resin) and optimal accessibility of the available binding sites. At the same time, minimal leakage of nanobodies is ensured also under harsh denaturing and/or reducing conditions. In contrast to conventional immunoprecipitations, the eluates of both ALFA selectors will not contain large amounts of co-eluted antibody fragments. Due to the combination of high capacity, minimal leakage and extraordinary low non-specific protein adsorption, both ALFA Selector resins allow for clean and highly specific immunoprecipitations. Selector resins are compatible not only with physiological buffers but also with high stringency buffers (see product-specific compatibility charts) and reducing agents.

Products

• N1510: ALFA SelectorPE, 2000 µl 50% slurry (1000 µl resin) + 1 mg elution peptide
• N1511: ALFA SelectorST, 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
• Acidic elution buffer: 0.1 M Glycin/HCl pH 2.2, 150 mM NaCl
• Neutralization buffer: 1M Tris pH 8.5
• 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 (analytical scale)

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 ALFA 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 ALFA 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. Wash beads with 1 ml lysis buffer and centrifuge for 1 min at 1000 x g.
11. Remove Supernatant.
12. Repeat steps 10 and 11.
13. Remove bottom plug from Mini-Spin column and place column in a 2 ml reaction tube.
14. Resuspend beads in 200 μl lysis buffer, transfer suspension to Mini-Spin column.
15. Wash out beads sticking to tube with 200 μl lysis buffer and transfer to column.
16. Centrifuge column for 1 min at 1000 x g, discard flow-through.
17. Wash twice with 400 μl washing buffer and centrifuge for 1 min at 1000 x g.
18. Wash once with 400 μl TBS and centrifuge for 1 min at 3000 x g.
19. Attach bottom plug and place Mini-Spin column in a clean 1.5 ml reaction tube.

Elution

The ALFA Selector resins can be eluted by different methods.

Peptide elution under native conditions (ALFA SelectorPE only)
This elution mode is based on competition between the ALFA elution peptide present in the elution buffer and the ALFA-tagged target protein for available binding sites on the resin. To obtain convenient elution kinetics, peptide elution has to be performed at room temperature (22 - 25°C).

Acidic elution (ALFA SelectorPE / ALFA SelectorST)
ALFA-tagged target proteins can efficiently be eluted from both ALFA Selector variants at low pH with Acidic elution buffer.

Denaturing elution using SDS sample buffer (ALFA SelectorPE / ALFA SelectorST)
ALFA-tagged target proteins can be efficiently eluted from both ALFA Selector variants using SDS sample buffer at elevated temperatures.

Elution procedures

Peptide elution (ALFA SelectorPE only)

1. Prepare a 100x (20 mM) stock solution of ALFA elution peptide by solubilizing the ALFA elution peptide at 40 mg/mL in deionized water.
2. Dilute stock solution 1:100 in physiological buffer to obtain an elution buffer containing 200 µM ALFA elution peptide.
3. Add 5 column bed volumes (CBV) elution buffer to the resin.
4. Incubate for 20 min at room temperature with subtle shaking.
5. Remove the bottom plug and collect the eluate by centrifugation 1 min at 1000 x g.
6. Optional: For highest yields attach bottom plug and repeat steps 3 - 5 and combine eluates.
7. Take sample from elution for further analysis (elution fraction).
8. Analyze collected samples by SDS-PAGE.

Elution under acidic conditions (ALFA SelectorPE / ALFA SelectorST)

1. Add 2.5 column bed volumes (CBV) Acidic elution buffer to the resin.
2. Incubate for 2 min at RT with subtle shaking.
3. Remove the bottom plug and collect the eluate by centrifugation 1 min at 1000 x g.
4. Attach bottom plug and repeat steps 1 - 3 once and combine eluates.
5. Take sample from elution for further analysis (elution fraction).
6. Analyze collected samples by SDS-PAGE.

Denaturing elution using SDS sample buffer (ALFA SelectorPE / ALFA SelectorST)

1. Add 2.5 column bed volumes (CBV) SDS sample buffer pre-warmed to approx. 60°C to the resin.
2. Close Mini-Spin column with lid.
3. Incubate for 5 min at 80 - 90°C with subtle shaking.
4. Open lid and remove the bottom plug and collect the eluate by centrifugation 1 min at 1000 x g.
5. Attach bottom plug and repeat steps 2 - 4 once and combine eluates.
6. Take sample from elution for further analysis (elution fraction).
7. Analyze collected samples by SDS-PAGE.

Protocol for preparative purifications using gravity flow

1. Prepare native cell lysates according to established protocols.
2. Clear lysate by centrifugation for 10 min at > 14000 x g and 4°C.
3. Resuspend ALFA Selector resin and transfer slurry to a clean gravity flow column.
4. Equilibrate resin with 5 column bed volumes (CBV) of lysis buffer.

Binding in batch mode

1. Add equilibrated ALFA Selector resin to the cleared lysate.
2. Incubate 1 h at 4°C with rotation.
3. Sediment beads by centrifugation for 1 min at 1000 x g and 4°C.
4. Discard the supernatant and transfer beads into the empty gravity flow column.

Binding in column mode

1. Slowly pass cleared lysate over the equilibrated column and collect flow through.
2. Optional: Pass non-bound material (flow-through) once more over the column.

Elution procedures

Peptide elution under native conditions (ALFA SelectorPE only)

This elution mode is based on competition between the ALFA elution peptide present in the elution buffer and the ALFA-tagged target protein for available binding sites on the resin. To obtain convenient elution kinetics, peptide elution has to be performed at room temperature (22 - 25°C). Elution at 4°C is inefficient.

1. Prepare 500 µM (0.9 mg/mL) ALFA elution peptide in native buffer.
2. Add 1.2 CBV elution buffer (equilibrated to RT) prepared in step 1 on top of the resin.
3. Collect eluate fraction and incubate 10 - 15 min at RT.
4. Repeat steps 2 and 3 three times.
5. Analyze input material, non-bound material and eluate fractions by SDS-PAGE.

Acidic elution (ALFA SelectorPE / ALFA SelectorST)

ALFA-tagged target proteins can efficiently be eluted from both ALFA Selector variants at low pH with Acidic elution buffer.
Note: Please make sure that the target protein is compatible with acidic elution conditions. Acidic elution can be performed at room temperature or at 4°C.

1. Add 1.2 CBV Acidic elution buffer on top of the resin.
2. Collect eluate fraction and immediately neutralize by adding 1/10 volume neutralization buffer.
3. Incubate 1 - 2 min.
4. Repeat steps 1 - 2 three times.
5. Analyze input material, non-bound material and eluate fractions by SDS-PAGE.