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.

All experiments carried out in animals were approved by the Committee for Laboratory Animal Care and Use of the Autonomous National University of M&#233;xico (UNAM), under the CICUAL-Protocol number: FLC40-14. (CICUAL: &#34;Comit&#233; Institucional para el Cuidado y Uso de los Animales de Laboratorio del Instituto de Fisiolog&#236;a Celular, Universidad Nacional Aut&#242;noma de M&#233;xico&#34;).
1. Preparation of Embryo Protein Extract
<ol>
	<li>Collect 100 - 200 embryos for each conditio...

<ol>
	<li>L&#243;pez-Casillas, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+expression+of+the+membrane+proteoglycan+betaglycan,+a+component+of+the+TGF-&#946;+receptor+system.">Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-&#946; receptor system.</a> Cell. 67 (4), 785-795 (1991).</li><li>Cheifetz, S., Andres, J. L., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transforming+growth+factor-beta+receptor+type+III+is+a+membrane+proteoglycan.+Domain+structure+of+the+receptor.">The transforming growth factor-beta receptor type III is a membrane proteoglycan. Domain structure of the receptor.</a> J. Biol. Chem. 263 (32), 16984-16991 (1988).</li><li>Kamaid, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Betaglycan+knock-down+causes+embryonic+angiogenesis+defects+in+zebrafish.">Betaglycan knock-down causes embryonic angiogenesis defects in zebrafish.</a> Genesis. 53, 583-603 (2015).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rapid+and+Sensitive+Method+for+the+Quantitation+of+Microgram+Quantities+of+Protein+Utilizing+the+Principle+of+Protein-Dye+Binding.">A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Cheifetz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+transforming+growth+factor-b+receptor+subsets+as+determinants+of+cellular+responsiveness+to+three+TGF-b+isoforms.">Distinct transforming growth factor-b receptor subsets as determinants of cellular responsiveness to three TGF-b isoforms.</a> J. Biol. Chem. 265, 20533-20538 (1990).</li><li>L&#243;pez-Casillas, F., Wrana, J. L., Massagu&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Betaglycan+presents+ligand+to+the+TGF&#946;+signaling+receptor.">Betaglycan presents ligand to the TGF&#946; signaling receptor.</a> Cell. 73 (7), 1435-1444 (1993).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> PNAS USA. 76 (9), 4350-4354 (1979).</li><li>Maccari, F., Volpi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+specific+recognition+of+glycosaminoglycans+by+antibodies+after+their+separation+by+agarose+gel+electrophoresis+and+blotting+on+cetylpyridinium+chloride-treated+nitrocellulose+membranes.">Direct and specific recognition of glycosaminoglycans by antibodies after their separation by agarose gel electrophoresis and blotting on cetylpyridinium chloride-treated nitrocellulose membranes.</a> Electrophoresis. 24 (9), 1347-1352 (2003).</li><li>Volpi, N., Maccari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosaminoglycan+blotting+and+detection+after+electrophoresis+separation.">Glycosaminoglycan blotting and detection after electrophoresis separation.</a> Methods Mol Biol (Clifton, N.J). 1312, 119-127 (2015).</li><li>Guesdon, J. L., Ternynck, T., Avrameas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+avidin-biotin+interaction+in+immunoenzymatic+techniques.">The use of avidin-biotin interaction in immunoenzymatic techniques.</a> J Histochem Cytochem. 27 (8), 1131-1139 (1979).</li><li>Bratthauer, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+avidin-biotin+complex+(ABC)+method+and+other+avidin-biotin+binding+methods.">The avidin-biotin complex (ABC) method and other avidin-biotin binding methods.</a> Methods Mol Biol (Clifton, N.J). 588, 257-270 (2010).</li><li>Heimer, R., Molinaro, L., Sampson, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+by+125I-cationized+cytochrome+c+of+proteoglycans+and+glycosaminoglycans+immobilized+on+unmodified+and+on+positively+charged+Nylon+66.">Detection by 125I-cationized cytochrome c of proteoglycans and glycosaminoglycans immobilized on unmodified and on positively charged Nylon 66.</a> Anal. Biochem. 165 (2), 448-455 (1987).</li><li>Das, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+radiolabeling+of+a+cell+surface+receptor+for+epidermal+growth+factor.">Specific radiolabeling of a cell surface receptor for epidermal growth factor.</a> PNAS USA. 74 (7), 2790-2794 (1977).</li><li>Massague, J., Guillette, B., Czech, M., Morgan, C., Bradshaw, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+nerve+growth+factor+receptor+protein+in+sympathetic+ganglia+membranes+by+affinity+labeling.">Identification of a nerve growth factor receptor protein in sympathetic ganglia membranes by affinity labeling.</a> J. Biol. Chem. 256 (18), 9419-9424 (1981).</li><li>Massague, J., Pilch, P. F., Czech, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+resolution+of+three+major+insulin+receptor+structures+with+unique+subunit+stoichiometries.">Electrophoretic resolution of three major insulin receptor structures with unique subunit stoichiometries.</a> PNAS USA. 77 (12), 7137-7141 (1980).</li><li>Hoosein, N. M., Gurd, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Glucagon+Receptors+in+Rat+Brain.">Identification of Glucagon Receptors in Rat Brain.</a> PNAS USA. 81, 4368-4372 (1984).</li></ol>

The use of Western blots with a specific antibody against a protein of interest is a valuable tool to study its expression7 during embryogenesis. However, immunoblotting of highly-glycosylated proteins has not been very successful due to their inefficient transfer and weak binding to nitrocellulose or PVDF membranes8,9.
Proteoglycans are a good example of this shortcoming, because of their covalently attached glycosaminoglycan chains (GAG) that are negatively charged and ...

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.

<table border="0" cellpadding="0" cellspacing="0" width="670">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Disuccinimidyl suberate (DSS)</td>
			<td style="width:168px;">ThermoFisher Scientific</td>
			<td style="width:119px;">21555</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Protein G Sephraose 4 Fast Flow</td>
			<td style="width:168px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">17-0618-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Gel Dryer Model 583&#160;</td>
			<td style="width:168px;">BIO-RAD</td>
			<td style="width:119px;">1651745</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Typhoon 9400</td>
			<td style="width:168px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">63-0055-78</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cobra II Auto gamma counter</td>
			<td style="width:168px;">Packard</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Exposure Cassette</td>
			<td style="width:168px;">Molecular Dynamics</td>
			<td style="width:119px;">63-0035-44</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Na2HPO4</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">K2HPO4</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3246</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CH4O</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">9070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C2H4O2</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">9508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CH2O</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">2106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">L4509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">ED</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">C3306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaHCO3</td>
			<td style="width:168px;">Fisher Scientific</td>
			<td style="width:119px;">S233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PMSF</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7626</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Crystal Sea Marine Mix</td>
			<td style="width:168px;">Marine Enterprises International</td>
			<td style="width:119px;">http://www.meisalt.com/Crystal-Sea-Marinemix</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 1 shows a representative result obtained with AFLIP. Signals in Lane 1 come from the 125I-ligand covalently linked to either the zebrafish betaglycan core protein (BG core, below the 150 kDa marker) or the BG core that has been processed to its proteoglycan form by attachment of glycosaminoglycans (GAG, smear ranging from 170 kDa to the top of the gel). This pattern of migration, a sharp core protein plus a smeared proteoglycan (due to heterogeneity in t...

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.

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

The overall goal of this procedure is to detect low abundance growth factor receptors in zebrafish embryos by combining the selectivity and sensitivity of affinity labeling of the chosen receptor by its radioactive ligand. This method can help answer questions in developmental biology fields such as receptor expression levels and their biochemical properties. This method which we have nicknamed AFLIP extend a powerful descriptive qualities of affinity labeling, a well-established cell biology technique to zebrafish developmental studies.To begin this procedure, collect 100 to 200 embryos for each condition and place them in petri dishes containing fish water. Then manually dechorionate the embryos using fine-tipped forceps and avoid using pronase or any other protease throughout the protocol as it may digest the targeted receptors. Next, place the embryos in a 1.5-milliliter microfuge tube and wash them twice with 1X PBS.Then add 500 microliters of deyolking buffer. Release most of the yolk by gently pipetting the embryos in the solution up and down about 30 to 40 times. For 72 HPF and 48 HPF embryos, use blue and yellow pipette tips respectively.Yellow tips may need to be slightly cut to avoid destroying the embryos. A successful yolk release can be checked by observing the embryos under the microscope. Removal of yolk must be as complete as possible as yolk lipids and proteins may interfere with subsequent ligand or antibody binding steps.After that, collect the embryos by centrifugation at 600 x g for 15 seconds. Carefully discard the supernatant using a pipette. Next, wash the embryos twice with 500 microliters of washing buffer by gently vortexing them at the lowest speed.Then collect the embryos by centrifugation at 600 x g for 15 seconds. From here onwards, continue the procedure at four degrees Celsius. Discard the supernatant carefully using a pipette.Afterward, we suspend the embryos in 350 microliters of lysis buffer and homogenize them using a plastic pestle. Then incubate the lysed embryos for 30 minutes with agitation on a test tube rocker at four degrees Celsius. Next, centrifuge the lysed embryos at 11, 000 x g for 15 minutes in order to remove the insoluble debris.Following that, transfer the cleared supernatant to a fresh tube. Determine the total protein by the Bradford protein assay and plotty calibration curve. In this step, place 400 to 500 micrograms of total embryo protein in a 1.5 milliliter microfuge tube and dilute it to one microgram per microliter with buffer 1.Next, add Iodine-125 label TGF-beta II stock to reach a final concentration of 150 picomolar and incubate with agitation on a test tube rocker for two hours at four degrees Celsius. After that, add undiluted antibody against the receptor of interest in order to reach a 1:100 dilution. Then continue the incubation at four degrees Celsius for another two hours or overnight.After incubation, add 50 microliters of suitable immunoglobulin binding beads and incubate for 50 minutes with agitation on a test tube rocker at four degrees Celsius. Then recover the beads by microcentrifugation at 11, 000 x g for 20 seconds and discard the supernatant in an appropriate radioactive trash container. Subsequently, wash the IP beads three times with one milliliter of IP wash buffer by vortexing at microcentrifugation at 11, 000 x g for 20 seconds each time.Then resuspend the IP beads in 250 microliters of IP wash buffer. Prepare fresh DSS solution and add 1.5 microliters of it to the sample. Following this, incubate the sample for 15 minutes at four degrees Celsius with agitation.In order to quench the cross-linking reaction, add 500 microliters of IP wash buffer supplemented with enough tryst chloride stock at pH 7.4 to reach 25 millimolar tryst chloride. Next, centrifuge at 11, 00 x g for 20 seconds to collect IP beads. Afterward, discard the supernatant and resuspend the IP beads in 30 microliters of reducing laemmli buffer.Next, boil the sample at 94 degrees Celsius for five minutes before SDS-PAGE gel electrophoresis and then analyze the dried gels in a phosphorimeter or by conventional autoradiography. This figure shows the representative results obtained with AFLIP. Total protein extracted from the embryos at 72 hpf and 48 hpf were subjected to Iodine-125 TGF-beta II affinity labeling followed by IP with rabbit anti-zebrafish BG or rabbit anti-rat BG as a control.In the autoradiography, BG can be detected without GAG or as a glycosaminoglycan-modified BG.This figure shows how AFLIP can be used to gauge the expression levels of a given receptor in embryos subjected to an experimental manipulation, in this case, the decrease of betaglycan to the morpholino injection. Lane one shows the level of TGF beta receptor III in a 72 hpf wild-type embryo, while lane two shows the effect of administering seven nanograms of a morpholino directed to the exon 2-intron 2 boundary of the ZFPG primary transcript. Note that the BG downregulation obtained with this morpholino is not reproduced by its mismatched control.These results confirmed that the phenotype obtained with this morpholino was specific to knockdown of the TGF beta receptor III as described before. The success of AFLIP for detecting a growth factor or cytokine receptors depends on the availability of a radiolabeled ligand that can be bound and cross-linked to its receptor. A good antibody for immunoprecipitation is also require.Once mastered, this technique can be done in two days if it is performed properly. Since AFLIP avoids the transfer step of the western blot, it allows for an enhanced detection of heavily glycosylated receptors. And we have seen here for the proteoglycan betaglycan which transfer very inefficiently in western blot Following this procedure, other methods like enzymatic digestion can be performed in order to describe additional post-translational modification as long as they are revealed by electrophoretic techniques.After watching this video, you should have a good understanding of how AFLIP can help you detect low abundant receptors in several fish embryos by affinity labeling linked to immunoprecipitation. Finally, don't forget that working with Iodine-125 must be done with care and adequate protection. Good luck with your experiments.

affinity-labeling-detection-endogenous-receptors-from-zebrafish

Research

JoVE Journal

Developmental Biology

5.5K 次观看.  Universidad Nacional Autónoma de México. The overall goal of this procedure is to detect low abundance growth factor receptors in zebrafish embryos by combining the selectivity and sensitivity of affinity labeling of the chosen receptor by its radioactive ligand. This method can help answer questions in developmental biology fields such as receptor expression levels and their biochemical properties. This method which we have nicknamed AFLIP extend a powerful descriptive qualities of affinity labeling, a well-established cell biology...