PMID- 36571722
OWN - NLM
STAT- MEDLINE
DCOM- 20221228
LR  - 20230106
IS  - 2691-1299 (Electronic)
IS  - 2691-1299 (Linking)
VI  - 2
IP  - 12
DP  - 2022 Dec
TI  - NanoTag Nanobody Tools for Drosophila In Vitro and In Vivo Studies.
PG  - e628
LID - 10.1002/cpz1.628 [doi]
AB  - Nanobodies have emerged as powerful protein-binding tools to uncover protein 
      functions. Using functionalized protein binders, proteins of interest can be 
      visualized, degraded, delocalized, or post-translationally modified in vivo. We 
      recently reported the use of two short peptide tags, 10-aa 127D01 and 14-aa 
      VHH05, and their corresponding nanobodies, Nb127D01 and NbVHH05, for both in 
      vitro and in vivo studies in Drosophila. Here, we provide detailed protocols for 
      nanobody production and for visualization of proteins of interest in either fixed 
      or live samples. In addition, we include protocols for endogenous protein tagging 
      using CRISPR-mediated genome engineering. © 2022 Wiley Periodicals LLC. Basic 
      Protocol 1: Nanobody production in S2 cells Basic Protocol 2: Nanobody expression 
      and purification in bacterial cells Basic Protocol 3: Immunostaining with 
      nanobodies Basic Protocol 4: Immunoblotting with nanobodies Basic Protocol 5: 
      Immunoprecipitation with nanobodies prepared from S2 cells Basic Protocol 6: 
      Immunoprecipitation with nanobodies prepared from bacteria Basic Protocol 7: 
      NbVHH05 and Nb127D01 used as chromobodies Basic Protocol 8: NanoTag trap as a 
      method to alter protein localization Support Protocol: CRISPR-mediated tagging of 
      endogenous genes with NanoTags.
CI  - © 2022 Wiley Periodicals LLC.
FAU - Kim, Ah-Ram
AU  - Kim AR
AD  - Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, 
      Massachusetts.
FAU - Xu, Jun
AU  - Xu J
AD  - Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, 
      Massachusetts.
AD  - CAS Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center 
      for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant 
      Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China.
FAU - Cheloha, Ross
AU  - Cheloha R
AD  - Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts.
FAU - Mohr, Stephanie E
AU  - Mohr SE
AD  - Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, 
      Massachusetts.
FAU - Zirin, Jonathan
AU  - Zirin J
AD  - Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, 
      Massachusetts.
FAU - Ploegh, Hidde L
AU  - Ploegh HL
AD  - Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts.
FAU - Perrimon, Norbert
AU  - Perrimon N
AD  - Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, 
      Massachusetts.
AD  - Howard Hughes Medical Institute, Boston, Massachusetts.
LA  - eng
GR  - P41 GM132087/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PL  - United States
TA  - Curr Protoc
JT  - Current protocols
JID - 101773894
RN  - 0 (Single-Domain Antibodies)
SB  - IM
MH  - Animals
MH  - *Single-Domain Antibodies/genetics/metabolism
MH  - Drosophila/metabolism
MH  - Protein Binding/genetics
MH  - Protein Transport
PMC - PMC9811555
MID - NIHMS1851725
OTO - NOTNLM
OT  - CRISPR
OT  - Drosophila
OT  - NanoTag
OT  - knock-in
OT  - nanobody
COIS- CONFLICT OF INTEREST STATEMENT The authors declare that they have no conflict of 
      interest.
EDAT- 2022/12/27 06:00
MHDA- 2022/12/29 06:00
PMCR- 2023/12/01
CRDT- 2022/12/26 11:22
PHST- 2023/12/01 00:00 [pmc-release]
PHST- 2022/12/26 11:22 [entrez]
PHST- 2022/12/27 06:00 [pubmed]
PHST- 2022/12/29 06:00 [medline]
AID - 10.1002/cpz1.628 [doi]
PST - ppublish
SO  - Curr Protoc. 2022 Dec;2(12):e628. doi: 10.1002/cpz1.628.

PMID- 35076390
OWN - NLM
STAT- MEDLINE
DCOM- 20220224
LR  - 20220224
IS  - 2050-084X (Electronic)
IS  - 2050-084X (Linking)
VI  - 11
DP  - 2022 Jan 25
TI  - Protein visualization and manipulation in Drosophila through the use of epitope 
      tags recognized by nanobodies.
LID - 10.7554/eLife.74326 [doi]
LID - e74326
AB  - Expansion of the available repertoire of reagents for visualization and 
      manipulation of proteins will help understand their function. Short epitope tags 
      linked to proteins of interest and recognized by existing binders such as 
      nanobodies facilitate protein studies by obviating the need to isolate new 
      antibodies directed against them. Nanobodies have several advantages over 
      conventional antibodies, as they can be expressed and used as tools for 
      visualization and manipulation of proteins in vivo. Here, we characterize two 
      short (<15aa) NanoTag epitopes, 127D01 and VHH05, and their corresponding 
      high-affinity nanobodies. We demonstrate their use in Drosophila for in vivo 
      protein detection and re-localization, direct and indirect immunofluorescence, 
      immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated 
      gene targeting provides a straightforward approach to tagging endogenous proteins 
      with the NanoTags. Single copies of the NanoTags, regardless of their location, 
      suffice for detection. This versatile and validated toolbox of tags and 
      nanobodies will serve as a resource for a wide array of applications, including 
      functional studies in Drosophila and beyond.
CI  - © 2022, Xu et al.
FAU - Xu, Jun
AU  - Xu J
AUID- ORCID: 0000-0002-7963-0253
AD  - Department of Genetics, Harvard Medical School, Boston, United States.
FAU - Kim, Ah-Ram
AU  - Kim AR
AUID- ORCID: 0000-0001-9597-6759
AD  - Department of Genetics, Harvard Medical School, Boston, United States.
FAU - Cheloha, Ross W
AU  - Cheloha RW
AD  - Boston Children's Hospital and Harvard Medical School, Boston, United States.
FAU - Fischer, Fabian A
AU  - Fischer FA
AD  - Boston Children's Hospital and Harvard Medical School, Boston, United States.
FAU - Li, Joshua Shing Shun
AU  - Li JSS
AD  - Department of Genetics, Harvard Medical School, Boston, United States.
FAU - Feng, Yuan
AU  - Feng Y
AD  - Department of Genetics, Harvard Medical School, Boston, United States.
FAU - Stoneburner, Emily
AU  - Stoneburner E
AD  - Department of Genetics, Harvard Medical School, Boston, United States.
FAU - Binari, Richard
AU  - Binari R
AD  - Department of Genetics, Harvard Medical School, Boston, United States.
FAU - Mohr, Stephanie E
AU  - Mohr SE
AUID- ORCID: 0000-0001-9639-7708
AD  - Department of Genetics, Harvard Medical School, Boston, United States.
AD  - Drosophila RNAi Screening Center, Harvard Medical School, Boston, United States.
FAU - Zirin, Jonathan
AU  - Zirin J
AD  - Department of Genetics, Harvard Medical School, Boston, United States.
AD  - Drosophila RNAi Screening Center, Harvard Medical School, Boston, United States.
FAU - Ploegh, Hidde L
AU  - Ploegh HL
AD  - Boston Children's Hospital and Harvard Medical School, Boston, United States.
FAU - Perrimon, Norbert
AU  - Perrimon N
AUID- ORCID: 0000-0001-7542-472X
AD  - Department of Genetics, Harvard Medical School, Boston, United States.
AD  - Drosophila RNAi Screening Center, Harvard Medical School, Boston, United States.
AD  - Howard Hughes Medical Institute, Boston, United States.
LA  - eng
GR  - P41 GM132087/GM/NIGMS NIH HHS/United States
GR  - HHMI/Howard Hughes Medical Institute/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20220125
PL  - England
TA  - Elife
JT  - eLife
JID - 101579614
RN  - 0 (Drosophila Proteins)
RN  - 0 (Epitopes)
RN  - 0 (Single-Domain Antibodies)
SB  - IM
MH  - Animals
MH  - Drosophila Proteins/*immunology
MH  - Drosophila melanogaster/*immunology
MH  - Epitopes/*immunology
MH  - Single-Domain Antibodies/*metabolism
PMC - PMC8853664
OTO - NOTNLM
OT  - D. melanogaster
OT  - Drosophila
OT  - biochemistry
OT  - chemical biology
OT  - genetics
OT  - genomics
OT  - nanobody
OT  - protein labeling
OT  - protein manipulation
OT  - short epitope tag
COIS- JX, AK, RC, FF, JL, YF, ES, RB, SM, JZ, HP, NP No competing interests declared
EDAT- 2022/01/26 06:00
MHDA- 2022/02/25 06:00
CRDT- 2022/01/25 12:19
PHST- 2021/09/29 00:00 [received]
PHST- 2022/01/24 00:00 [accepted]
PHST- 2022/01/26 06:00 [pubmed]
PHST- 2022/02/25 06:00 [medline]
PHST- 2022/01/25 12:19 [entrez]
AID - 74326 [pii]
AID - 10.7554/eLife.74326 [doi]
PST - epublish
SO  - Elife. 2022 Jan 25;11:e74326. doi: 10.7554/eLife.74326.