Developing high-quality mouse monoclonal antibodies for neuroscience research - approaches, perspectives and opportunities.
High-quality antibodies (Abs) are critical to neuroscience research, as they remain the primary affinity proteomics reagent used to label and capture endogenously expressed protein targets in the nervous system. As in other fields, neuroscientists are frequently confronted with inaccurate and irreproducible Ab-based results and/or reporting. The UC Davis/NIH NeuroMab Facility was created with the mission of addressing the unmet need for high-quality Abs in neuroscience research by applying a unique approach to generate and validate mouse monoclonal antibodies (mAbs) optimized for use against mammalian brain (i.e., NeuroMabs). Here we describe our methodology of multi-step mAb screening focused on identifying mAbs exhibiting efficacy and specificity in labeling mammalian brain samples. We provide examples from NeuroMab screens, and from the subsequent specialized validation of those selected as NeuroMabs. We highlight the particular challenges and considerations of determining specificity for brain immunolabeling. We also describe why our emphasis on extensive validation of large numbers of candidates by immunoblotting and immunohistochemistry against brain samples is essential for identifying those that exhibit efficacy and specificity in those applications to become NeuroMabs. We describe the special attention given to candidates with less common non-IgG1 IgG subclasses that can facilitate simultaneous multiplex labeling with subclass-specific secondary antibodies. We detail our recent use of recombinant cloning of NeuroMabs as a method to archive all NeuroMabs, to unambiguously define NeuroMabs at the DNA sequence level, and to re-engineer IgG1 NeuroMabs to less common IgG subclasses to facilitate their use in multiplex labeling. Finally, we provide suggestions to facilitate Ab development and use, as to design, execution and interpretation of Ab-based neuroscience experiments. Reproducibility in neuroscience research will improve with enhanced Ab validation, unambiguous identification of Abs used in published experiments, and end user proficiency in Ab-based assays.
Pubmed ID: 26644354 RIS Download
Research resources used in this publication
- VAChT transporter antibody (RRID:AB_2532047) (antibody)
- GABA-A-R-Alpha2 (RRID:AB_2336904) (antibody)
- SynCAM4 (RRID:AB_10673109) (antibody)
- VAChT transporter antibody (RRID:AB_2532047) (antibody)
- Anti-RGS14 Antibody (RRID:AB_10698026) (antibody)
- Pan-Nav1 Na+ channel (RRID:AB_2491079) (antibody)
- Ab1-pan-MAGUK, anti-membrane-associated guanylate kinase (RRID:AB_10698179) (antibody)
- Pan-KChIP potassium channel (RRID:AB_2132595) (antibody)
- Navbeta2 sodium channel (RRID:AB_2315895) (antibody)
- Navbeta4 sodium channel (RRID:AB_10673578) (antibody)
- Mortalin/GRP75 (RRID:AB_10674108) (antibody)
- KChIP4 K+ channel (RRID:AB_2491080) (antibody)
- HCN2 ion channel (RRID:AB_10672304) (antibody)
- KCC2 transporter (RRID:AB_10697875) (antibody)
- Anti-GluA1/GluR1 Glutamate Receptor Antibody (RRID:AB_2315839) (antibody)
- GABA(A)R, Alpha5 (RRID:AB_2491075) (antibody)
- GABA(A)R, Pi antibody (RRID:AB_2493092) (antibody)
- GABA-A-R-Alpha2 (RRID:AB_2336904) (antibody)
- Ankyrin-R (RRID:AB_2336901) (antibody)
- Anti-Ankyrin-G (Staining) Antibody (RRID:AB_10697718) (antibody)
- Anti-IL17RB polyclonal antibody (RRID:AB_1234567) (antibody)
Additional research tools detected in this publication
Antibodies used in this publication
- VAChT transporter antibody (RRID:AB_2532047)
- SynCAM4 (RRID:AB_10673109)
- Anti-RGS14 Antibody (RRID:AB_10698026)
- Pan-Nav1 Na+ channel (RRID:AB_2491079)
- Ab1-pan-MAGUK, anti-membrane-associated guanylate kinase (RRID:AB_10698179)
- Pan-KChIP potassium channel (RRID:AB_2132595)
- Navbeta4 sodium channel (RRID:AB_10673578)
- Navbeta2 sodium channel (RRID:AB_2315895)
- Mortalin/GRP75 (RRID:AB_10674108)
- KChIP4 K+ channel (RRID:AB_2491080)
- KCC2 transporter (RRID:AB_10697875)
- HCN2 ion channel (RRID:AB_10672304)
- Anti-GluA1/GluR1 Glutamate Receptor Antibody (RRID:AB_2315839)
- GABA(A)R, Pi antibody (RRID:AB_2493092)
- GABA(A)R, Alpha5 (RRID:AB_2491075)
- GABA-A-R-Alpha2 (RRID:AB_2336904)
- Ankyrin-R (RRID:AB_2336901)
- Anti-Ankyrin-G (Staining) Antibody (RRID:AB_10697718)
- Anti-SynCAM4 Antibody (RRID:AB_10676101)
- Anti-RGS14 Antibody (RRID:AB_2179931)
- Anti-Pan-Nav1 Sodium Channel Antibody (RRID:AB_2491098)
- Anti-Pan-MAGUK Antibody (RRID:AB_10673115)
- Anti-Pan-KChIP K+ Channel Antibody (RRID:AB_10673406)
- Anti-Navbeta4 Na+ Channel Antibody (RRID:AB_2301402)
- Anti-Navbeta2 Na+ Channel Antibody (RRID:AB_2315896)
- Anti-Mortalin/GRP75 Antibody (RRID:AB_2120479)
- Anti-KChIP4 K+ channel Antibody (RRID:AB_2493100)
- Anti-KCC2 Antibody (RRID:AB_10672851)
- Anti-HCN2 Antibody (RRID:AB_2279449)
- Anti-GluA1/GluR1 Glutamate Receptor Antibody (RRID:AB_2315840)
- Anti-GABA-A-R-Pi Antibody (RRID:AB_2532051)
- Anti-GABA-A-R-Alpha5 Antibody (RRID:AB_2491187)
- Anti-Ankyrin-R Antibody (RRID:AB_2491109)
- Anti-Ankyrin-G (Staining) Antibody (RRID:AB_10673030)
- Anti-IL17RB polyclonal antibody (RRID:AB_1234567)
Associated grants
- Agency: NINDS NIH HHS, United States
Id: R24 NS092991 - Agency: NINDS NIH HHS, United States
Id: U24 NS050606
Publication data is provided by the National Library of Medicine ® and PubMed ®. Data is retrieved from PubMed ® on a weekly schedule. For terms and conditions see the National Library of Medicine Terms and Conditions.
This is a list of tools and resources that we have found mentioned in this publication.
Neuroscience Information Framework (tool)
RRID:SCR_002894
Framework for identifying, locating, relating, accessing, integrating, and analyzing information from neuroscience research. Users can search for and add neuroscience-related resources at NIF portal and receive and RRID to track and cite resources within scientific manuscripts.
View all literature mentionsResource Identification Portal (tool)
RRID:SCR_004098
Portal providing identifiers for Antibodies, Model Organisms, and Tools (software, databases, services) created in support of the Resource Identification Initiative, which aims to promote research resource identification, discovery, and reuse. The portal offers a central location for obtaining and exploring Research Resource Identifiers (RRIDs) - persistent and unique identifiers for referencing a research resource. A critical goal of the RII is the widespread adoption of RRIDs to cite resources in the biomedical literature and other places that reference their generation or use. RRIDs use established community identifiers where they exist, and are cross-referenced in their system where more than one identifier exists for a single resource.
View all literature mentionsJournal of Comparative Neurology Antibody database (tool)
RRID:SCR_006470
THIS RESOURCE IS NO LONGER IN SERVICE. Documented on May 4th,2023. It was integrated with Antibody Registry. The JCN antibody database is a listing of all antibodies used in JCN papers from 2006 onward. The catalog numbers and vendor information is included for all antibodies listed, and with a new collaboration with NIF''''s AntibodyRegistry, a unique identifier is also listed for each antibody. The Journal of Comparative Neurology requires rigorous characterization for all antibodies that are used in JCN papers. The antibodies in the The Journal of Comparative Neurology antibody database have in nearly all cases been described and characterized adequately according to the provided guidelines. This information can be used to identify a particular target immunohistochemically or to design an experiment using the antibody information. If you are looking for an antibody to identify a particular target immunohistochemically, this list is a good place to begin your search. We suggest you then look up the paper in which the antibody was used, to make sure that it will meet your needs and to verify its characterization. (The characterization of antibodies in JCN papers often goes well beyond the material published by the manufacturer, so that examining this information before you order an antibody can be very useful.) While we do not guarantee that these antibodies will identify only the intended target (that is a function of the actual experiment and controls), this is the most carefully verified list of antibodies that we are aware of, and we wanted to share this resource with our readers and authors.
View all literature mentionsVAChT transporter antibody (antibody)
RRID:AB_2532047
This monoclonal targets VAChT transporter
View all literature mentionsAnti-RGS14 Antibody (antibody)
RRID:AB_10698026
This monoclonal targets RGS14
View all literature mentionsPan-Nav1 Na+ channel (antibody)
RRID:AB_2491079
This monoclonal targets
View all literature mentionsAb1-pan-MAGUK, anti-membrane-associated guanylate kinase (antibody)
RRID:AB_10698179
This monoclonal targets Pan-MAGUK
View all literature mentionsPan-KChIP potassium channel (antibody)
RRID:AB_2132595
This monoclonal targets Pan-KChIP potassium channel
View all literature mentionsNavbeta4 sodium channel (antibody)
RRID:AB_10673578
This monoclonal targets Navbeta4 sodium channel
View all literature mentionsNavbeta2 sodium channel (antibody)
RRID:AB_2315895
This monoclonal targets Navbeta2 sodium channel
View all literature mentionsMortalin/GRP75 (antibody)
RRID:AB_10674108
This monoclonal targets Mortalin/GRP75
View all literature mentionsKCC2 transporter (antibody)
RRID:AB_10697875
This monoclonal targets KCC2 transporter
View all literature mentionsHCN2 ion channel (antibody)
RRID:AB_10672304
This monoclonal targets HCN2 ion channel
View all literature mentionsAnti-GluA1/GluR1 Glutamate Receptor Antibody (antibody)
RRID:AB_2315839
This monoclonal targets GluA1/GluN1 glutamate receptor
View all literature mentionsGABA(A)R, Pi antibody (antibody)
RRID:AB_2493092
This monoclonal targets GABA(A)R, Pi
View all literature mentionsGABA-A-R-Alpha2 (antibody)
RRID:AB_2336904
This monoclonal targets GABA-A-R-Alpha2
View all literature mentionsAnti-Ankyrin-G (Staining) Antibody (antibody)
RRID:AB_10697718
This monoclonal targets Ankyrin-G (staining) scaffold protein
View all literature mentionsAnti-SynCAM4 Antibody (antibody)
RRID:AB_10676101
This monoclonal targets SynCAM4
View all literature mentionsAnti-RGS14 Antibody (antibody)
RRID:AB_2179931
This monoclonal targets RGS14
View all literature mentionsAnti-Pan-Nav1 Sodium Channel Antibody (antibody)
RRID:AB_2491098
This monoclonal targets Pan-Nav1 sodium channel
View all literature mentionsAnti-Pan-MAGUK Antibody (antibody)
RRID:AB_10673115
This monoclonal targets Pan-MAGUK
View all literature mentionsAnti-Pan-KChIP K+ Channel Antibody (antibody)
RRID:AB_10673406
This monoclonal targets Pan-KChIP K+ channel
View all literature mentionsAnti-Navbeta4 Na+ Channel Antibody (antibody)
RRID:AB_2301402
This monoclonal targets Navbeta4 Na+ channel
View all literature mentionsAnti-Navbeta2 Na+ Channel Antibody (antibody)
RRID:AB_2315896
This monoclonal targets Navbeta2 Na+ channel
View all literature mentionsAnti-Mortalin/GRP75 Antibody (antibody)
RRID:AB_2120479
This monoclonal targets Mortalin/GRP75
View all literature mentionsAnti-KChIP4 K+ channel Antibody (antibody)
RRID:AB_2493100
This monoclonal targets KChIP4 K+ channel
View all literature mentionsAnti-KCC2 Antibody (antibody)
RRID:AB_10672851
This monoclonal targets KCC2
View all literature mentionsAnti-HCN2 Antibody (antibody)
RRID:AB_2279449
This monoclonal targets HCN2
View all literature mentionsAnti-GluA1/GluR1 Glutamate Receptor Antibody (antibody)
RRID:AB_2315840
This monoclonal targets GluA1/GluR1 glutamate receptor
View all literature mentionsAnti-GABA-A-R-Pi Antibody (antibody)
RRID:AB_2532051
This monoclonal targets GABA-A-R-Pi
View all literature mentionsAnti-GABA-A-R-Alpha5 Antibody (antibody)
RRID:AB_2491187
This monoclonal targets GABA-A-R-Alpha5
View all literature mentionsAnti-Ankyrin-R Antibody (antibody)
RRID:AB_2491109
This monoclonal targets Ankyrin-R
View all literature mentionsAnti-Ankyrin-G (Staining) Antibody (antibody)
RRID:AB_10673030
This monoclonal targets Ankyrin-G (Staining)
View all literature mentionsAnti-IL17RB polyclonal antibody (antibody)
RRID:AB_1234567
This polyclonal targets IL17RB
View all literature mentionsVAChT transporter antibody (antibody)
RRID:AB_2532047
This monoclonal targets VAChT transporter
View all literature mentionsGABA-A-R-Alpha2 (antibody)
RRID:AB_2336904
This monoclonal targets GABA-A-R-Alpha2
View all literature mentionsVAChT transporter antibody (antibody)
RRID:AB_2532047
This monoclonal targets VAChT transporter
View all literature mentionsAnti-RGS14 Antibody (antibody)
RRID:AB_10698026
This monoclonal targets RGS14
View all literature mentionsPan-Nav1 Na+ channel (antibody)
RRID:AB_2491079
This monoclonal targets
View all literature mentionsAb1-pan-MAGUK, anti-membrane-associated guanylate kinase (antibody)
RRID:AB_10698179
This monoclonal targets Pan-MAGUK
View all literature mentionsPan-KChIP potassium channel (antibody)
RRID:AB_2132595
This monoclonal targets Pan-KChIP potassium channel
View all literature mentionsNavbeta2 sodium channel (antibody)
RRID:AB_2315895
This monoclonal targets Navbeta2 sodium channel
View all literature mentionsNavbeta4 sodium channel (antibody)
RRID:AB_10673578
This monoclonal targets Navbeta4 sodium channel
View all literature mentionsMortalin/GRP75 (antibody)
RRID:AB_10674108
This monoclonal targets Mortalin/GRP75
View all literature mentionsHCN2 ion channel (antibody)
RRID:AB_10672304
This monoclonal targets HCN2 ion channel
View all literature mentionsKCC2 transporter (antibody)
RRID:AB_10697875
This monoclonal targets KCC2 transporter
View all literature mentionsAnti-GluA1/GluR1 Glutamate Receptor Antibody (antibody)
RRID:AB_2315839
This monoclonal targets GluA1/GluN1 glutamate receptor
View all literature mentionsGABA(A)R, Pi antibody (antibody)
RRID:AB_2493092
This monoclonal targets GABA(A)R, Pi
View all literature mentionsGABA-A-R-Alpha2 (antibody)
RRID:AB_2336904
This monoclonal targets GABA-A-R-Alpha2
View all literature mentionsAnti-Ankyrin-G (Staining) Antibody (antibody)
RRID:AB_10697718
This monoclonal targets Ankyrin-G (staining) scaffold protein
View all literature mentionsAnti-IL17RB polyclonal antibody (antibody)
RRID:AB_1234567
This polyclonal targets IL17RB
View all literature mentions
