Western blot analysis of N- and C-terminal Spot-tagged GFP added to HEK- 293T cell lysate. Detection with Spot-tag® antibody [28A5] (28a5, ChromoTek) 1:5,000 and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 488 [CTK0103, CTK0104] (sms1AF488-1, ChromoTek) 1:5,000. Western blot membrane was incubated simultaneously with the primary antibody and Nano-Secondary® (one-step staining).
Western blot analysis of C-terminal Spot-tagged GFP added to HEK-293T cell lysate. Detection with Spot-tag® antibody [28A5] (28a5, ChromoTek) 1:5,000 and anti-mouse secondary antibody HRP 1:1,000.
Western blot analysis of different cell lysates +/- 50 ng C-terminal Spot-tagged GFP: human (HEK-293T), yeast (S.cerevisiae), insect (High Five™), bacteria (E.coli) and plant (A.thaliana). Detection with Spot-tag® antibody [28A5] (28a5, ChromoTek) 1:5,000 and Nano-Secondary® alpaca anti-mouse IgG1, recombinant VHH, Alexa Fluor® 488 [CTK0103, CTK0104] (sms1AF488-1, ChromoTek) 1:5,000. Western blot membrane was incubated simultaneously with the primary antibody and Nano-Secondary® (one-step staining).
ELISA detection of N- and C-terminal Spot-tagged GFP with Spot-tag® antibody [28A5] (28a5, ChromoTek). N- and C-terminal Spot-tagged GFP was coated on a microtiter plate, blocked and detected with 1 nM Spot-tag® antibody [28A5] (28a5, ChromoTek) and anti-mouse IgG antibody AP.
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.
Spot-Traps (7 total products)
Spot-Labels (5 total products)
