Research ResourceBiochemistry

The Myc tag monoclonal antibody 9E10 displays highly variable epitope recognition dependent on neighboring sequence context

See allHide authors and affiliations

Science Signaling  28 Jan 2020:
Vol. 13, Issue 616, eaax9730
DOI: 10.1126/scisignal.aax9730
  • Fig. 1 Recognition of Myc-tagged PP2A Aα subunit by clone 9E10 is strongly influenced by the epitope sequence context.

    (A) Immunogen sequence corresponding to amino acids 408 to 439 of the human c-Myc used for generating clone 9E10. Clone 4A6 was raised against amino acids 410 to 419 (underlined). (B) Immunoblotting of lysates from NIH3T3 mouse fibroblasts either transfected with empty vector or expressing the indicated versions of Myc-tagged PP2A Aα subunit using the Myc-specific antibody 9E10 and a PP2A A–specific polyclonal antibody. One membrane was immunoblotted for β-tubulin as loading control. (C) Immunoblotting of lysates from NIH3T3 mouse fibroblasts either transfected with empty vector or expressing the indicated versions of Myc-tagged PP2A Aα subunit using antibody 4A6. The same amounts of the identical lysates that were used in (B) were loaded for this blot. Blots are representative of n = 4 independent experiments.

  • Fig. 2 Clone 9E10 shows much higher signal variability than clone 4A6 on peptide microarray.

    Peptides corresponding to the Myc epitope tag in the contexts MEQKLISEEDLXXKR or MEQKLISEEDLXXST, in which the XX positions were permuted to all 20 amino acids, were incubated with the indicated antibody on PEPperCHIP Custom Peptide Microarrays. Fluorescence intensity values (y axis, log scale) are plotted for 800 double-substitution permuted peptides (x axis). Twenty peptides were not detected by clone 9E10 and are therefore not represented in the log diagram. Primary data are shown in table S2. a.u., arbitrary units.

  • Fig. 3 Amino acid content C terminal to the Myc tag strongly influences the detection of tagged proteins by clone 9E10 in Western blotting.

    (A) Immunoblotting of lysates from bacteria expressing six versions of an N-terminal Myc-tagged PP2A B55α subunit fragment in different C-terminal sequence contexts [NGST, IGST, IAST, LRKR, TRKR, and L(GS)3] using the PP2A B55α–specific monoclonal antibody 2G9 as reference antibody and the Myc tag–specific antibodies, clone 9E10 and clone 4A6. Representative images of n = 8 independent LI-COR Western blot analyses are shown. (B) Quantification of fluorescence signal intensities adjusted to the 2G9 signals, which represent the amounts of Myc-tagged PP2A B55α subunit fragment, shown as average ± SD from eight Western blot replicates (n = 8). The average NGST context signal was arbitrarily set to 1, and values are shown relative to NGST. Statistical significance was calculated with a one-way ANOVA + post hoc Tukey’s HSD test. P values relative to NGST are depicted as ****P ≤ 0.001.

  • Fig. 4 Antibody binding to Myc tag on Western blots depends on tag position and sequence context.

    (A) Immunoblotting of lysates from bacteria expressing the four indicated different context versions of an Myc-tagged PP2A B55α subunit fragment using the indicated antibodies. Representative images of n = 8 independent LI-COR Western blot analyses are shown. Two antibodies recognizing PP2A B55α (clone 2G9 and rabbit polyclonal Δ237) were used as reference antibodies. (B) Quantification of fluorescence signal intensities adjusted to the 2G9 signals, which represent the amounts of Myc-tagged PP2A B55α subunit fragment, shown as average ± SD from eight Western blot replicates (n = 8). Statistical significance of signal differences between contexts was calculated separately for each antibody with a one-way ANOVA + post hoc Tukey’s HSD test. P values are shown relative to the pCMV-N value for the respective antibody: *P ≤ 0.05, ****P ≤ 0.001.

  • Table 1 Protein sequences of six N-terminal Myc-tagged PP2A Bα subunit fragments.

    The core Myc tag epitope is underlined, and the different sequence contexts are shown in red. The epitope of clone 2G9 is shown in bold.

    Myc-NGST-B55α 301 to 447
    MEQKLISEEDLNGSTMTRDYLSVKVWDLNMENRPVETYQVHEYLRSKLCSLYE
    NDCIFDKFECCWNGSDSVVMTGSYNNFFRMFDRNTKRDITLEASRENNKPR
    TVLKPRKVCASGKRKKDEISVDSLDFNKKILHTAWHPKENIIAVATTNNLYI
    FQDKVN
    Myc-IGST-B55α 301 to 447
    MEQKLISEEDLIGSTMTRDYLSVKVWDLNMENRPVETYQVHEYLRSKLCSLYE
    NDCIFDKFECCWNGSDSVVMTGSYNNFFRMFDRNTKRDITLEASRENNKPR
    TVLKPRKVCASGKRKKDEISVDSLDFNKKILHTAWHPKENIIAVATTNNLYI
    FQDKVN
    Myc-IAST-B55α 301 to 447
    MEQKLISEEDLIASTMTRDYLSVKVWDLNMENRPVETYQVHEYLRSKLCSLYE
    NDCIFDKFECCWNGSDSVVMTGSYNNFFRMFDRNTKRDITLEASRENNKPR
    TVLKPRKVCASGKRKKDEISVDSLDFNKKILHTAWHPKENIIAVATTNNLYI
    FQDKVN
    Myc-LRKR-B55α 301 to 447
    MEQKLISEEDLLRKRMTRDYLSVKVWDLNMENRPVETYQVHEYLRSKLCSLYE
    NDCIFDKFECCWNGSDSVVMTGSYNNFFRMFDRNTKRDITLEASRENNKPR
    TVLKPRKVCASGKRKKDEISVDSLDFNKKILHTAWHPKENIIAVATTNNLYI
    FQDKVN
    Myc-TRKR-B55α 301 to 447
    MEQKLISEEDLTRKRMTRDYLSVKVWDLNMENRPVETYQVHEYLRSKLCSLYE
    NDCIFDKFECCWNGSDSVVMTGSYNNFFRMFDRNTKRDITLEASRENNKPR
    TVLKPRKVCASGKRKKDEISVDSLDFNKKILHTAWHPKENIIAVATTNNLYI
    FQDKVN
    Myc-L(GS)3-B55α 301 to 447
    MEQKLISEEDLLGSGSGSMTRDYLSVKVWDLNMENRPVETYQVHEYLRSKLCS
    LYENDCIFDKFECCWNGSDSVVMTGSYNNFFRMFDRNTKRDITLEASRENN
    KPRTVLKPRKVCASGKRKKDEISVDSLDFNKKILHTAWHPKENIIAVATTNN
    LYIFQDKVN
  • Table 2 Protein sequences of four Myc-tagged PP2A Bα subunit fragments.

    The core Myc tag epitope is underlined, and the different sequence contexts are shown in red. The epitope of clone 2G9 is shown in bold.

    pCMV-Myc-N-B55α 301 to 447
    MEQKLISEEDLLMAMEARMTRDYLSVKVWDLNMENRPVETYQVHEYLRSKL
    CSLYENDCIFDKFECCWNGSDSVVMTGSYNNFFRMFDRNTKRDITLEASR
    ENNKPRTVLKPRKVCASGKRKKDEISVDSLDFNKKILHTAWHPKENIIAVA
    TTNNLYIFQDKVN
    pCMV-Myc-C-B55α 301 to 447
    MTRDYLSVKVWDLNMENRPVETYQVHEYLRSKLCSLYENDCIFDKFECC
    WNGSDSVVMTGSYNNFFRMFDRNTKRDITLEASRENNKPRTVLKPRKV
    CASGKRKKDEISVDSLDFNKKILHTAWHPKENIIAVATTNNLYIFQDKVN
    EISRGTGEQKLISEEDL
    pCruz-Myc-B55α 301 to 447
    MGSEEQKLISEEDLEFSTAAAMTRDYLSVKVWDLNMENRPVETYQVHEY
    LRSKLCSLYENDCIFDKFECCWNGSDSVVMTGSYNNFFRMFDRNTKRD
    ITLEASRENNKPRTVLKPRKVCASGKRKKDEISVDSLDFNKKILHTAWH
    PKENIIAVATTNNLYIFQDKVN
    pcDNA3.1/Myc-His B-B55α 301 to 447
    MTRDYLSVKVWDLNMENRPVETYQVHEYLRSKLCSLYENDCIFDKFECC
    WNGSDSVVMTGSYNNFFRMFDRNTKRDITLEASRENNKPRTVLKPRKV
    CASGKRKKDEISVDSLDFNKKILHTAWHPKENIIAVATTNNLYIFQDKVN
    SLEGPRFEQKLISEEDL

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/616/eaax9730/DC1

    Fig. S1. Clone 9E10 is greatly diminished in immunoprecipitation of Myc–NGST–PP2A Aα.

    Fig. S2. Clone 9E10 shows sequence context bias toward 6xMyc-tagged proteins.

    Table S1. 9E10 shows a larger signal variability than 4A6 in a single-substitution scan of Myc peptide contexts NGST or LRKR.

    Table S2. 9E10 shows a larger signal variability than 4A6 in a double-substitution scan of the first two amino acid positions C terminal to Myc peptide.

    Table S3. Highest- and lowest-scoring Myc context peptides in a double-substitution scan for clones 4A6 and 9E10.

  • The PDF file includes:

    • Fig. S1. Clone 9E10 is greatly diminished in immunoprecipitation of Myc–NGST–PP2A Aα.
    • Fig. S2. Clone 9E10 shows sequence context bias toward 6xMyc-tagged proteins.
    • Legends for tables S1 to S3

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). 9E10 shows a larger signal variability than 4A6 in a single-substitution scan of Myc peptide contexts NGST or LRKR.
    • Table S2 (Microsoft Excel format). 9E10 shows a larger signal variability than 4A6 in a double-substitution scan of the first two amino acid positions C terminal to Myc peptide.
    • Table S3 (Microsoft Excel format). Highest- and lowest-scoring Myc context peptides in a double-substitution scan for clones 4A6 and 9E10.

Stay Connected to Science Signaling

Navigate This Article