Summary

تقارب كشف وسمها الذاتية المستقبلات من الزرد الأجنة

Published: August 31, 2016
doi:

Summary

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

Abstract

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-β receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-β2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

Introduction

Specific detection of proteins expressed during embryonic development is required to validate expression profiles obtained by measuring their cognate mRNAs with RT-PCR or in situ hybridization (ISH). This is commonly achieved by a western blot of embryo extracts followed by detection with specific antibodies. However, this approach is hard to apply to proteins that are in very low abundance, or that have properties that hamper their quantitative transfer during their blotting. Betaglycan, also known as the type III transforming growth factor β (TGF-β) receptor (TGFBR3), is an example of these difficulties. TGFBR3 is a part time membrane proteoglycan that binds TGF-β through its core protein1, with notably higher affinity for the isoform TGF-β2, a property that distinguishes it from any other TGF-β binding protein2. TGFBR3 in the zebrafish is expressed from 8 hpf on, reaching a maximum by 72 hpf, as detected by RT-PCR of its mRNA3.

However, despite the availability of a very specific antibody3, every attempt to detect its translated product by western blot proved unsuccessful. Reckoning that TGFBR3’s proteoglycan nature, as well as putative low abundance may be accountable for this failure, a detection method, AFLIP, which takes advantage of TGFBR3 high affinity for TGF-β2 was devised. In this method a protein extract from pooled embryos is allowed to specifically bind 125I-labeled TGF-β2 and the receptor-ligand complexes are purified by immunoprecipitation and cross-linked before separation by SDS-PAGE. The migration patterns observed by autoradiography of the gels revealed the presence and nature of the labeled receptor species. This approach combines the ligand specificity of affinity labeling with immunoprecipitation by specific antibodies, increasing detection range, avoiding the inefficient transfer blotting of TGFBR3. Due to its inherent properties, the AFLIP assay is not a quantitative assay but can be used to confidently gauge relative experimental differences in the analyzed receptor.

Protocol

تمت الموافقة على جميع التجارب التي أجريت على الحيوانات من قبل لجنة رعاية الحيوان المعملية واستخدام الجامعة الوطنية المستقلة في المكسيك (UNAM)، تحت رقم CICUAL-البروتوكول: FLC40-14. (CICUAL: "COMITE Institucional الفقرة شرم CUIDADO ذ الالتزام بتعميم الخدمات دي لوس Animales دي ابوراتوريو ديل معهد…

Representative Results

ويبين الشكل 1 نتيجة ممثل حصلت مع AFLIP. إشارات في حارة 1 تأتي من 125 I-يجند مرتبطة تساهميا إما إلى الزرد البروتين betaglycan الأساسية (BG الأساسية، تحت علامة كيلو دالتون 150) أو جوهر BG التي تم تجهيزها لشكله بروتيوغليكان عن طريق الحجز من الجليكوسامي?…

Discussion

استخدام البقع الغربية مع الأجسام المضادة المحددة ضد بروتين من الفائدة هو أداة قيمة لدراسة التعبير عنها 7 خلال مرحلة التطور الجنيني. ومع ذلك، لم يكن immunoblotting من البروتينات عالية الغليكوزيلاتي ناجحة للغاية نظرا لنقلهم عدم الكفاءة وضعف ملزم لالنيتروسليلوز أو PVDF ا…

Declarações

The authors have nothing to disclose.

Acknowledgements

The authors thank Gilberto Morales for fish care and maintenance, and Drs. Claudia Rivera and Hector Malagòn (IFC-UNAM Animal Facility) for their help in rabbit immunization. This work was supported by grants from CONACYT 131226 and PAPIIT-DGAPA-UNAM IN204916.

Materials

Disuccinimidyl suberate (DSS) ThermoFisher Scientific 21555
Protein G Sephraose 4 Fast Flow GE Healthcare Life Sciences 17-0618-01
Gel Dryer Model 583  BIO-RAD 1651745
Typhoon 9400 GE Healthcare Life Sciences 63-0055-78
Cobra II Auto gamma counter Packard
Exposure Cassette Molecular Dynamics 63-0035-44
NaCl J.T. Baker 3624
KCl J.T. Baker 3040
Na2HPO4 J.T. Baker 3828
K2HPO4 J.T. Baker 3246
CH4O J.T. Baker 9070
C2H4O2 J.T. Baker 9508
CH2O J.T. Baker 2106
SDS Sigma-Aldrich L4509
EDTA Sigma-Aldrich ED
Triton X-100 Sigma-Aldrich T9284
CaCl2 Sigma-Aldrich C3306
NaHCO3 Fisher Scientific S233
PMSF Sigma-Aldrich P7626
Crystal Sea Marine Mix Marine Enterprises International http://www.meisalt.com/Crystal-Sea-Marinemix

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Molina-Villa, T., Mendoza, V., López-Casillas, F. Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos. J. Vis. Exp. (114), e54405, doi:10.3791/54405 (2016).

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