SDS-PAGE of EGFP: 10 % acrylamide gel; 1 µg of EGFP has been loaded. Detection: InstantBlue

Normalized EGFP fluorescence excitation and emission spectra.

Model fig of Spot peptide.

Purification of Spot-tagged GFP with Spot-Cap from HEK293T cell lysate in gravity flow column format. Elution 1-6: 6 fractions à 2 CVs Spot-peptide (100 μM) in PBS.

Workflow of Spot-Cap purification.

Spot-Cap has a higher selectivity than anti-DYKDDDDK resin. While Spot-Cap is optimized for minimal contamination of host cell proteins, the anti-DYKDDDDK resin binds significant amounts of mammalian proteins. Incubation of control HEK293T cell lysate expressing no tagged protein with Spot-Cap or anti-DYKDDDDK resin. Elution with 100 μM Spot-peptide or 0.1 g/L DYKDDDDK-peptide. Similar results were obtained for yeast, bacteria, and insect cell lysates (data not shown).

The elution efficiency of Spot-Cap is constant even after 4 regeneration cycles. Elution of Spot-tagged PCNA with Spot-peptide. R0: Reference (elution fraction of Spot-PCNA without regeneration); R1-R4: Elution fraction of Spot-PCNA after 1, 2, 3, and 4 regeneration cycles with 100 mM glycine pH 2.0.

Purification of Spot-tagged GFP with Spot-Cap from HEK293T cell lysate in batch format. Elution: 6 fractions à 2 BVs Spot-peptide (100 μM) in PBS.

Application of Spot-tag related products.

DYKDDDDK Fab-Trap™ was used for immunoprecipitation of Flag®-tagged protein from HEK293T cell lysate. During elution with 3xDYKDDDDK-peptide (fp-1) the enriched Flag® fusion protein is released from DYKDDDDK Fab-Trap™. Western blot was probed with DYKDDDDK tag Polyclonal antibody (Binds to FLAG® tag epitope) (Proteintech, 20543-1-AP) and Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa Fluor® 488 [CTK0101, CTK0102] (srbAF488-1). L: Prestained protein marker (Proteintech, PL00001), I: Input, FT: Flow-Through, E: Elution.

The HA-Trap Agarose was used to immunoprecipitate HA-PCNA fusion protein from HEK293T cells. HA-PCNA protein was released from the trap through a two-step competitive elution with HA-peptide. Samples from the Input (I), Flow-Through (F), 1st elution (E1), 2nd elution (E2), and residual (R) fractions were analyzed through WB. PCNA Monoclonal Antibody (60097-1-Ig) and Multi-rAb HRP-Goat Anti-Mouse Recombinant Secondary Antibody (RGAM001) were used in the WB analysis. Note: PCNA forms trimers resulting in co-elution of endogenous PCNA proteins with HA-tagged PCNA.

The HA-Trap Magnetic Agarose was used to immunoprecipitate HA-PCNA fusion protein from HEK293T cells. HA-PCNA protein was released from the trap through a two-step competitive elution utizling HA-peptide. Samples from the Input (I), Flow-Through (F), 1st elution (E1), 2nd elution (E2), and residual (R) fractions were analyzed through WB. PCNA Monoclonal Antibody (60097-1-Ig) and Multi-rAb HRP-Goat Anti-Mouse Recombinant Secondary Antibody (RGAM001) were used in the WB analysis. Note: PCNA forms timers resulting in co-elution of endogenous PCNA proteins with HA-tagged PCNA.

The HA-Trap Magnetic Particles M-270 was used to immunoprecipitate HA-PCNA fusion protein from HEK293T cells. HA-PCNA protein was released from the trap through a two-step competitive elution utizling HA-peptide. Samples from the Input (I), Flow-Through (F), 1st elution (E1), 2nd elution (E2), and residual (R) fractions were analyzed through WB. PCNA Monoclonal Antibody (60097-1-Ig) and Multi-rAb HRP-Goat Anti-Mouse Recombinant Secondary Antibody (RGAM001) were used in the WB analysis. Note: PCNA forms timers resulting in co-elution of endogenous PCNA proteins with HA-tagged PCNA.