All steps for ELISA tests were performed at ambient temperature in TBS and TBS-KCl with 0.05% (v/v) Tween-20. 5′-biotinylated DNA (100 nM) was coated via neutravidin for 1 h. IMAC-purified DARPins (50 nM) or 1:10 diluted crude extracts were incubated for 40 min. An anti RGS-His antibody (Qiagen, Germany) and an anti-mouse antibody alkaline phosphatase conjugate (Sigma) were used for detection.
Anti rgs his antibody
Manufactured by Qiagen
Sourced in Germany
The Anti-RGS-His antibody is a recombinant antibody that binds to the RGS-His tag, which is commonly used for the purification and detection of recombinant proteins. This antibody can be used in various applications such as Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assays (ELISA).
Automatically generated - may contain errors
4 protocols using anti rgs his antibody
1
ELISA Assay for DARPin Binding
2
Extracellular IL-13Rα1 Protein Expression
3
Visualizing RGS-His Protein via Western Blot
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β-mercaptoethanol was added to pull-down eluates to 5%. Samples were heated 20 min at 85°C and equivalent volumes were loaded on a 15% SDS-polyacrylamide gel. Proteins were visualized by Western blot analysis with anti-RGS-His antibody (Qiagen), ECL anti-mouse horseradish peroxidase-linked antibody (GE Healthcare) and ECL Western Blotting Detection reagent (Amersham).
4
Tagging Dbp2p with HTBH in Yeast
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Construction of plasmid pFA6a-HTB containing the HTB-tagged Dbp2p was described previously (Tagwerker et al. 2006 (link)). pFA6a-HTB was then used to generate the plasmid pFA6a-HTBH (BTP16). The HIS-tag was added by PCR amplification with primers HIS F and HIS R, generating pFA6a-HTBH (BTP16). This plasmid was then used as a template for PCR-mediated homologous recombination to tag DBP2 with HTBH in the yeast genome. Expression of Dbp2p-HTBH was verified by western blot using anti-RGS-His antibody (Qiagen). Growth characteristics of the DBP2-HTBH strain were examined by serial dilution experiments, compared to the wild-type strain (Supplemental Fig. S1A ). Cells were grown in YP + 2%D to mid-log phase, harvested by centrifugation and re-suspended in 1× TE to an OD600 nm = 0.5. Fivefold serial dilutions were prepared, spotted on YP + 2%D plates and grown from 2 to 7 d at temperatures ranging from 16°C to 37°C (Supplemental Fig. S1A ). Generation of the dbp2Δ strain has been described previously (Cloutier et al. 2012 (link)).
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