Figure 4.
Evaluating the specific binding of RNAP2 Ser2ph-mintbody to phosphopeptides in vitro. RNAP2 Ser2ph-mintbody was expressed in E. coli as the His-tag form, purified through an Ni column, treated with enterokinase to remove His-tag, and further purified. (A) Purified proteins were separated on an SDS-polyacrylamide gel and stained with Coomassie Blue. Positions of size standards are indicated on the left. (B) ELISA plates that were coated with synthetic peptides conjugated with BSA, incubated with a dilution series of purified RNAP2 Ser2ph-mintbody, the parental 42B3 antibody (IgG), and control antibodies specific for RNAP2 Ser2ph (CMA602) and RNAP2 Ser5ph (CMA603). After incubations with peroxidase-conjugated anti-GFP (for RNAP2 Ser2ph-mintbody) or antimouse IgG (for mAb) and then with o-phenylenediamine, absorbance at 490 nm was measured. RNAP2 Ser2ph-mintbody reacted with peptides containing Ser2ph. Source data are available for this figure: SourceData F4.

Evaluating the specific binding of RNAP2 Ser2ph-mintbody to phosphopeptides in vitro. RNAP2 Ser2ph-mintbody was expressed in E. coli as the His-tag form, purified through an Ni column, treated with enterokinase to remove His-tag, and further purified. (A) Purified proteins were separated on an SDS-polyacrylamide gel and stained with Coomassie Blue. Positions of size standards are indicated on the left. (B) ELISA plates that were coated with synthetic peptides conjugated with BSA, incubated with a dilution series of purified RNAP2 Ser2ph-mintbody, the parental 42B3 antibody (IgG), and control antibodies specific for RNAP2 Ser2ph (CMA602) and RNAP2 Ser5ph (CMA603). After incubations with peroxidase-conjugated anti-GFP (for RNAP2 Ser2ph-mintbody) or antimouse IgG (for mAb) and then with o-phenylenediamine, absorbance at 490 nm was measured. RNAP2 Ser2ph-mintbody reacted with peptides containing Ser2ph. Source data are available for this figure: SourceData F4.

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