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.
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.
We recommend using Mini-Spin columns for immunoprecipitation experiments. An alternative batch protocol is found on the following page.
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.
Peptide elution (ALFA SelectorPE only)
Elution under acidic conditions (ALFA SelectorPE / ALFA SelectorST)
Denaturing elution using SDS sample buffer (ALFA SelectorPE / ALFA SelectorST)
Binding in batch mode
Binding in column mode
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.
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.