Fig. 2: Detection of HP-NAP by anti-FLAG M2 antibody.
From: Helical structure motifs made searchable for functional peptide design
a Detection of HP-NAP by anti-FLAG M2 antibody using recombinant HP-NAP-based ELISA. Recombinant HP-NAP was purified by a small-scale DEAE Sephadex negative mode batch chromatography. Purified HP-NAP as an antigen was coated on an ELISA plate as described in “Methods.” The anti-FLAG M2 antibody, its corresponding mouse IgG antibody, and the antibody 16F4 against HP-NAP, as a positive control, were subjected to recombinant HP-NAP-based ELISA. The result is expressed as absorbance at OD450 nm. Data are presented as mean ± standard deviation (S.D.) of one experiment in duplicate (black dots are individual results). Similar results were obtained in two independent experiments. The difference between IgG isotype and any of the other two groups are statistically significant (p = 0.00017 between M2 and IgG; p = 0.00004 between 16F4 and IgG) according to Student’s t test (two-tailed), and the source data are provided as a Source data file. b Detection of recombinant HP-NAP by anti-FLAG M2 antibody using western blot analysis. Recombinant HP-NAP was purified by two consecutive gel-filtration chromatography. MBP-tagged HP-NAPR77-E116 and MBP were partially purified by the MBPTrap HP column using the ÄKTA Purifier. These purified proteins, 1 μg each, were subjected to SDS-PAGE on a 15% gel and western blot analysis with anti-FLAG M2 antibody and the antibody 16F4. Molecular masses (M) in kDa are indicated in the left of the blots and the gel. Similar results were obtained in two independent experiments. c The sequence of HP-NAP. Note that “DYKYLE,” highlighted in the red box, matches the pattern D-Y-K-x-x-[DE] and is included in the R77-E116 stretch. The image is downloaded from PDB website (code:1JI4) with minor modifications.
