For a poster with detailed information on the ALFA System and its applications click here.
The ALFA-tag is a novel, rationally designed epitope tag (SRLEEELRRRLTE) that forms a small and stable α-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts.
The flanking proline residues are not part of the recognition sequence but may aid in spatial separation of the tag from the protein of interest.
The ALFA-tag has the broadest spectrum of applications in life sciences compared to any other tag. We developed a nanobody binding the ALFA-tag with ~26 pM affinity, which is ideally suited not only for super-resolution microscopy, immunoprecipitations, and Western blotting but also allows detection of proteins within living cells. This nanobody can be conjugated to virtually all kinds of fluorophores, enzymes or other biomolecules. Check out the FluoTag-X2 anti-ALFA in our webshop.
For immunoprecipitations we offer two types of ALFA Selector resins. Both ALFA Selectors are based on covalently coupled nanobodies that specifically recognize ALFA-tagged targets. ALFA Selector ST (for Super-Tight) offers the highest possible affinity for ALFA-tagged targets, which can be eluted under acidic or denaturing conditions. ALFA Selector PE (for Peptide Elution) displays an engineered nanobody with lower affinity (Kd ~11 nM), which is optimized for peptide elution under physiological conditions. Both Selector resins feature low non-specific protein adsorption and therefore allowing clean and meaningful immunoprecipitations. Due to the covalent and oriented immobilization, the immobilized nanobodies will stay on the resin even when using harsh denaturing and/or reducing conditions for elution. In contrast to conventional immunoprecipitations, eluates from both ALFA Selectors will therefore be free from large amounts of antibody fragments.
HeLa mock lysate prepared in PBS was mixed with recombinant purified ALFA-tagged GFP substrate (right lanes). The lysate/substrate mixture was incubated with either ALFA SelectorPE (left panel) or ALFA SelectorST (right panel). After washing with PBS, bound proteins were eluted either with 200µM ALFA-peptide, glycin pH2.2 or SDS sample buffer (SDS). Peptide elution under native conditions is efficient when using ALFA SelectorPE. The target protein can be recovered from both ALFA Selector resins by elution with glycin or SDS sample buffer.
Note that the purity of the target protein eluted from both ALFA Selector resins using low pH or peptide (only ALFA SelectorPE) is superior to the input material, which has been purified by a 2-step chromatography process involving affinity chromatography with proteolytic release and gel filtration.