Fine Mutational Analysis of 2B8 and 3H7 Tag Epitopes with Corresponding Specific Monoclonal Antibodies

Abstract

Bacteriophytochromes are phytochrome-like light-sensing photoreceptors that use biliverdin as a chromophore. To study the biochemical properties of the Deinococcus radiodurans bacteriophytochrome (DrBphP) protein, two anti-DrBphP mouse monoclonal antibodies (2B8 and 3H7) were generated. Their specific epitopes were identified in our previous report. We present here fine epitope mapping of these two antibodies by using truncation and substitution of original epitope sequences in order to identify minimized epitope peptides. The previously reported original epitope sequences for 2B8 and 3H7 were truncated from both sides. Our analysis showed that the minimal peptide sequence lengths for 2B8 and 3H7 antibodies were nine amino acids (RDPLPFFPP) and six amino acids (PGEIEE), respectively. We further characterized these peptides in order to investigate their reactivity after single deletion and single substitution of the original peptides. We found that single-substituted 2B8 epitope (RDPLPAFPP) and dual-substituted 3H7 epitope (PGEIAD) showed significantly increased reactivity. These two antibodies with high reactivity for the short modified peptide sequences are valueble for developing new peptide tags for protein research.

Arabidopsis RING E3 ubiquitin ligase JUL1 participates in ABA‐mediated microtubule depolymerization, stomatal closure, and tolerance response to drought stress

“…Wild-type, jul1, and atairp3/log2-2 plants were grown for 2 weeks on soil under long-day conditions (16:8 h light/dark photocycle) and then subjected to dehydration stress as described by Lee et al (2009). A cut rosette water loss assay was performed as described in a previous study (Kim et al, 2016).…”

Flashlight into the Function of Unannotated C11orf52 using Affinity Purification Mass Spectrometry

“…These plasmids were transiently transfected in HEK 293T cells, and the cell lysates were immunoprecipitated with Myc, Flag, and 2B8 tags. We used the 2B8 tags developed from bacteriophytochromes ( Deinococcus radiodurans ), having only nine amino acids of “RD­PLP­FFPP,” with a length similar to that of 10 amino acids of Myc (“EQK­LIS­EEDL”) and 8 amino acids of Flag (“DYK­DDD­DK”). , Because the 2B8 tag is a proline-rich tag, it minimizes the charge effects and the altered secondary structure for nonspecific binding. Second, the immunopurified complexes were digested with trypsin following the S-trap digestion method.…”