Name: affinity labeling detection of endogenous receptors from zebrafish embryos
Uploaded: 2016-08-31T15:00:00
Description: Watch this Scientific Journal Video about Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos at JoVE.com

The authors thank Gilberto Morales for fish care and maintenance, and Drs. Claudia Rivera and Hector Malag&#242;n (IFC-UNAM Animal Facility) for their help in rabbit immunization. This work was supported by grants from CONACYT 131226 and PAPIIT-DGAPA-UNAM IN204916.

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: &quot;COMITE Institucional الفقرة شرم CUIDADO ذ الالتزام بتعميم الخدمات دي لوس Animales دي ابوراتوريو ديل معهد...

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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...

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 &#946; (TGF-&#946;) receptor (TGFBR3), is an example of these difficulties. TGFBR3 is a part time membrane proteoglycan that binds TGF-&#946; through its core protein1, with notably higher affinity for the isoform TGF-&#946;2, a property that distinguishes it from any other TGF-&#946; 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-&#946;2 was devised. In this method a protein extract from pooled embryos is allowed to specifically bind 125I-labeled TGF-&#946;2 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.

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affinity labeling detection of endogenous receptors from zebrafish embryos

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-&#946; 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-&#946;2-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.

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?...

Watch this Scientific Journal Video about Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos at JoVE.com

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

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.

تقارب كشف وسمها الذاتية المستقبلات من الزرد الأجنة

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Research

JoVE Journal

Developmental Biology

Mechanical Vessel Injury in Zebrafish Embryos

إصابة سفينة الميكانيكية في الزرد الأجنة

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

تطبيق للDNA محددة وصمة عار الميثيل الأخضر في بطاقات المواد الفلورية الأجنة

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

عزل وتوصيف الخلايا واحدة من اسماك الزرد الأجنة

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

جيل من التعايش الالتصاقي الزرد الأجنة التي كتبها فيوجن الجراحي لتطوير الأريمات

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

التلاعب الصيغة الصبغية من الزرد الأجنة مع الحرارة صدمة 2 العلاج

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

بيوسنسينغ موتور الخلايا العصبية الغشاء المحتملة في الأجنة الزرد الحية

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

تحريض نقص الأكسجة في الضفدع الحي والأجنة الزرد

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

التصور للنشاط الكهربائي الخلوية في الزرد الأجنة في وقت مبكر، والأورام

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

طريقه تركيب طبقات لتمديد المجهر البؤري المجهري لأجنه الزبرافيش الكاملة بفواصل زمنيه طويلة

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

كامل جبل المناعة الكيمياء في الأجنة حمار وحشي واليرقات

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...