Research in the Lowe-Krentz lab is supported by research grant HL54269 from the National Institutes of Health to LLK.

1. Design of a GFP-protein Construct
<ol>
	<li>Purchase, or design and build, a GFP-tagged construct based on the protein in question. 
	NOTE: For a purchased construct, standard vectors are available from commercial laboratories that include some or all of the following suggestions: For a membrane protein, select a C-terminal location of the GFP because it is less likely to interfere with membrane protein trafficking. Consider an extension between the gene of interest and the GFP if there is reason to believe the...

<ol>
	<li>Pugh, R. J., 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=Transmembrane+Protein+184A+Is+a+Receptor+Required+for+Vascular+Smooth+Muscle+Cell+Responses+to+Heparin.">Transmembrane Protein 184A Is a Receptor Required for Vascular Smooth Muscle Cell Responses to Heparin.</a> J Biol Chem. 291, 5326-5341 (2016).</li><li>Daher, W., 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=Identification+of+Toxoplasma+TgPH1,+a+pleckstrin+homology+domain-containing+protein+that+binds+to+the+phosphoinositide+PI(3,5)P.">Identification of Toxoplasma TgPH1, a pleckstrin homology domain-containing protein that binds to the phosphoinositide PI(3,5)P.</a> Mol Biochem Parasitol. , (2016).</li><li>Vit, O., 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=Large-scale+identification+of+membrane+proteins+based+on+analysis+of+trypsin-protected+transmembrane+segments.">Large-scale identification of membrane proteins based on analysis of trypsin-protected transmembrane segments.</a> J Proteomics. , (2016).</li><li>Attwood, M. 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=Topology+based+identification+and+comprehensive+classification+of+four-transmembrane+helix+containing+proteins+(4TMs)+in+the+human+genome.">Topology based identification and comprehensive classification of four-transmembrane helix containing proteins (4TMs) in the human genome.</a> BMC genomics. 17, 268 (2016).</li><li>Zou, Z., 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=Genome-Wide+Identification+of+Jatropha+curcas+Aquaporin+Genes+and+the+Comparative+Analysis+Provides+Insights+into+the+Gene+Family+Expansion+and+Evolution+in+Hevea+brasiliensis.">Genome-Wide Identification of Jatropha curcas Aquaporin Genes and the Comparative Analysis Provides Insights into the Gene Family Expansion and Evolution in Hevea brasiliensis.</a> Front Plant Sci. 7, 395 (2016).</li><li>Gilotti, A. C., 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=Heparin+responses+in+vascular+smooth+muscle+cells+involve+cGMP-dependent+protein+kinase+(PKG).">Heparin responses in vascular smooth muscle cells involve cGMP-dependent protein kinase (PKG).</a> J Cell Physiol. 229, 2142-2152 (2014).</li><li>Farwell, S. L., 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=Heparin+Decreases+in+Tumor+Necrosis+Factor+alpha+(TNFalpha)-induced+Endothelial+Stress+Responses+Require+Transmembrane+Protein+184A+and+Induction+of+Dual+Specificity+Phosphatase+1.">Heparin Decreases in Tumor Necrosis Factor alpha (TNFalpha)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1.</a> J Biol Chem. 291, 5342-5354 (2016).</li><li>Xu, D., Esko, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demystifying+heparan+sulfate-protein+interactions.">Demystifying heparan sulfate-protein interactions.</a> Annu Rev Biochem. 83, 129-157 (2014).</li><li>Chiodelli, P., Bugatti, A., Urbinati, C., Rusnati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin/Heparan+sulfate+proteoglycans+glycomic+interactome+in+angiogenesis:+biological+implications+and+therapeutical+use.">Heparin/Heparan sulfate proteoglycans glycomic interactome in angiogenesis: biological implications and therapeutical use.</a> Molecules. 20, 6342-6388 (2015).</li><li>Slee, J. B., Lowe-Krentz, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+realignment+and+cofilin+regulation+are+essential+for+barrier+integrity+during+shear+stress.">Actin realignment and cofilin regulation are essential for barrier integrity during shear stress.</a> J Cell Biochem. 114, 782-795 (2013).</li><li>Patton, W. 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=Identification+of+a+heparin-binding+protein+using+monoclonal+antibodies+that+block+heparin+binding+to+porcine+aortic+endothelial+cells.">Identification of a heparin-binding protein using monoclonal antibodies that block heparin binding to porcine aortic endothelial cells.</a> The Biochemical journal. 311, 461-469 (1995).</li><li>Doggett, T. M., Breslin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+actin+cytoskeleton+in+live+endothelial+cells+expressing+GFP-actin.">Study of the actin cytoskeleton in live endothelial cells expressing GFP-actin.</a> J Vis Exp. , (2011).</li><li>Skalamera, D., 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=Generation+of+a+genome+scale+lentiviral+vector+library+for+EF1alpha+promoter-driven+expression+of+human+ORFs+and+identification+of+human+genes+affecting+viral+titer.">Generation of a genome scale lentiviral vector library for EF1alpha promoter-driven expression of human ORFs and identification of human genes affecting viral titer.</a> PloS one. 7, 51733 (2012).</li><li>Castro, M., Nikolaev, V. O., Palm, D., Lohse, M. J., Vilardaga, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turn-on+switch+in+parathyroid+hormone+receptor+by+a+two-step+parathyroid+hormone+binding+mechanism.">Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism.</a> Proc Natl Acad Sci U S A. 102, 16084-16089 (2005).</li><li>Albertazzi, L., Arosio, D., Marchetti, L., Ricci, F., Beltram, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+FRET+analysis+with+the+EGFP-mCherry+fluorescent+protein+pair.">Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair.</a> Photochem Photobiol. 85, 287-297 (2009).</li><li>Wang, 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=Domain+organization+of+the+ATP-sensitive+potassium+channel+complex+examined+by+fluorescence+resonance+energy+transfer.">Domain organization of the ATP-sensitive potassium channel complex examined by fluorescence resonance energy transfer.</a> J Biol Chem. 288, 4378-4388 (2013).</li><li>Christiansen, E., Hudson, B. D., Hansen, A. H., Milligan, G., Ulven, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Characterization+of+a+Potent+Free+Fatty+Acid+Receptor+1+(FFA1)+Fluorescent+Tracer.">Development and Characterization of a Potent Free Fatty Acid Receptor 1 (FFA1) Fluorescent Tracer.</a> J Med Chem. 59, 4849-4858 (2016).</li><li>Chiang, C. F., Okou, D. T., Griffin, T. B., Verret, C. R., Green Williams, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein+rendered+susceptible+to+proteolysis:+positions+for+protease-sensitive+insertions.">fluorescent protein rendered susceptible to proteolysis: positions for protease-sensitive insertions.</a> Arch Biochem Biophys. 394, 229-235 (2001).</li></ol>

The authors have nothing to disclose.

The protocols reported here were designed to provide confirmatory evidence for the identification of TMEM184A as a heparin receptor in vascular cells1. Knockdown techniques are routinely used as one mechanism to confirm the identification of novel proteins. However, some functional loss after knockdown is typically not sufficient proof that a candidate protein is actually the correct receptor (or other functional protein). It is also important to have evidence that the candidate protein actually e...

Identification of candidate proteins for novel functions often depends upon affinity-based isolation protocols followed by partial sequence determination. Recent examples of newly identified proteins include transmembrane protein 184A (TMEM184A), a heparin receptor identified after heparin affinity interactions1, and TgPH1, a pleckstrin homology domain protein that binds phosphoinositide PI(3,5)P22. Other novel protein identification involves direct sequence analysis of peptides such as that by Vit, et al. who used transmembrane peptides to identify protein products from previously uncharacterized genes3. Similarly, identification of novel protein sequences can be accomplished using bioinformatics searching of previously characterized protein families such as identification of new 4TM proteins4. Examination of aquaporin family gene sequences has also yielded the identification of new members with novel functions5. After identification, analysis of protein function is typically a next step which can sometimes be examined using a specific assay of protein function such as in the aquaporin case.
When possible, function of a newly identified protein can be examined with specific enzymatic or similar in vitro function assays. Because many functions of novel proteins depend on complex interactions that occur only in intact cells or organisms, in vitro assays are not always effective. However, the in vivo assays must be designed in such a way that they depend on the gene sequence. In cell culture, and/or simple model organisms, knockdown can provide supporting evidence for the protein/function identification6. With novel proteins identified as noted above, it is often insufficient to simply knock down a protein to confirm function, and the design of in vivo functional assays that depend on gene sequence becomes important for the characterization of novel proteins.
The recent identification of TMEM184A as a heparin receptor (that modulates proliferation in vascular smooth muscle and inflammatory responses in endothelial cells) using affinity chromatography and MALDI MS1,7 provided an opportunity to develop a collection of assays after knockdown yielded results consistent with the identification. A recent review confirmed that heparin interacts specifically with many growth factors, their receptors, extracellular matrix components, cell adhesion receptors, and other proteins8. In the vascular system, heparin and heparan sulfate proteoglycans (containing heparan sulfate chains similar in structure to heparin) interact with several hundred proteins9. To functionally confirm that TMEM184A was involved with heparin uptake and binding, techniques that employed the gene construct for TMEM184A were developed. The present report includes a collection of assays based on a GFP-TMEM184A construct for use in confirming the identity of TMEM184A as a heparin receptor.

<table border="0" cellpadding="0" cellspacing="0" width="767">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GFP-TMEM184A construct</td>
			<td style="width:71px;">OriGene</td>
			<td style="width:119px;">RG213192</td>
			<td style="width:361px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Rhodamine-Heparin</td>
			<td style="width:71px;">Creative PEGWorks</td>
			<td style="width:119px;">HP-204</td>
			<td style="width:361px;">Light Sensitive</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fluorescein-Heparin</td>
			<td style="width:71px;">Creative PEGWorks</td>
			<td style="width:119px;">HP-201</td>
			<td style="width:361px;">Light Sensitive</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Mowiol</td>
			<td style="width:71px;">EMD Millipore</td>
			<td style="width:119px;">475904-100GM</td>
			<td style="width:361px;"></td>
		</tr>
		<tr height="141">
			<td height="141" style="height:141px;width:216px;">Paraformaldehyde (methanol free)</td>
			<td style="width:71px;">Thermo Sci Pierce Biotech, available through Fisher Scientific</td>
			<td style="width:119px;">PI28908 at Fisher</td>
			<td style="width:361px;">Use in Fume Hood</td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:216px;">Reacti-bind neutravidin plates (Avidin coated black 96 well dishes)</td>
			<td style="width:71px;">Thermo Sci Pierce Biotech, through Fisher Scientific</td>
			<td style="width:119px;">PI15510 at Fisher</td>
			<td style="width:361px;">Pay attention to shelf-life</td>
		</tr>
		<tr height="161">
			<td height="161" style="height:161px;width:216px;">Black 96 well plates</td>
			<td style="width:71px;">Corning Life Sciences Plastic, purchased through Fisher Scientific</td>
			<td style="width:119px;">064432 at Fisher</td>
			<td style="width:361px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">A7r5 vascular smooth muscle cell line</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">CRL 1444</td>
			<td style="width:361px;">Can be exchanged into MEM medium1</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">BAOEC bovine aortic endothelial cells</td>
			<td style="width:71px;">Cell Applications, Inc.</td>
			<td style="width:119px;">B304-05</td>
			<td style="width:361px;">Culture as recommended initially, can be exchanged into MEM medium for continuing culture1,7</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">BAOSMC bovine aortic smooth muscle cells</td>
			<td style="width:71px;">Cell Applications, Inc.</td>
			<td style="width:119px;">B354-05</td>
			<td style="width:361px;">Culture as recommended initially, can be exchanged into MEM medium for continuing culture1</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">RAOEC rat aortic endothelial cells</td>
			<td style="width:71px;">Cell Applications, Inc.</td>
			<td style="width:119px;">R304-05a</td>
			<td style="width:361px;">Culture as recommended initially, can be exchanged into MEM medium for continuing culture7</td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:216px;">Biotinylated anti-GFP</td>
			<td style="width:71px;">Thermo Sci Pierce Biotech, through Fisher Scientific</td>
			<td style="width:119px;">MA5-15256-BTIN</td>
			<td style="width:361px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptavidin-coated beads</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S1638</td>
			<td style="width:361px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">HeBS</td>
			<td style="width:71px;">Available from Bio-Rad</td>
			<td style="width:119px;"></td>
			<td style="width:361px;">Can be prepared in the lab.&#160; The pH is 6.8</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">TMEM184A antibody to the N-terminus</td>
			<td style="width:71px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc292006</td>
			<td style="width:361px;">Only known TMEM184A antibody to N-terminal region.</td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:216px;">TMEM184A antibody to the C-terminus</td>
			<td style="width:71px;">Obtained from ProSci Inc, Poway, CA&#160;</td>
			<td style="width:119px;">Pro Sci 5681</td>
			<td style="width:361px;">ProSci used in figure 1</td>
		</tr>
		<tr height="20">
			<td height="41" style="height:41px;width:216px;">GFP antibodies</td>
			<td style="width:71px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc9996</td>
			<td style="width:361px;">Used in figures 5</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Secondary antibodies, labeled with TRITC or Cy3</td>
			<td style="width:71px;">Jackson ImmunoResearch Laboratories, Inc, West Grove, PA</td>
			<td style="width:119px;">711 025 152 (donkey anti-rabbit, TRITC) 
			715 165 150 (donkey anti-mouse, Cy3)</td>
			<td style="width:361px;">Minimal cross-reactivity to minimize any non-specific staining.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">CHAPS</td>
			<td style="width:71px;">Purchased from Sigma</td>
			<td style="width:119px;">C5849</td>
			<td style="width:361px;">Note that this specific catalog number has been discontinued.&#160; Supplier will provide information regarding replacement.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Live imaging 35 mm dishes</td>
			<td style="width:71px;">MatTek (Ashland MA)</td>
			<td style="width:119px;">P35G-1.0 &#8211; 20 mm - C</td>
			<td style="width:361px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Confocal Microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">LSM 510 Meta with a 63X oil-immersion lens</td>
			<td style="width:361px;">Used for images and live-imaging in Figures 1, 2 and 3</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Confocal Microscope</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">C2+ confocal with a 60X oil-immersion lens</td>
			<td style="width:361px;">Used for images in Figure 5</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Confocal Microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Zeiss LSM 880 with a 63X oil-immersion lens</td>
			<td style="width:361px;">Used for images in Figure 2C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Electroporation equipment</td>
			<td style="width:71px;">Bio-Rad</td>
			<td style="width:119px;">Gene Pulser X-Cell System</td>
			<td style="width:361px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Electroporation cuvettes</td>
			<td style="width:71px;">Available from MidSci</td>
			<td style="width:119px;">EC2L</td>
			<td style="width:361px;">Can also be obtained from equipment supplier</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Plate reader</td>
			<td style="width:71px;">TECAN</td>
			<td style="width:119px;">TECAN Infinite&#174; m200 Pro plate reader</td>
			<td style="width:361px;">Readings in the middle of the wells rather than at the surface.</td>
		</tr>
		<tr height="40">
			<td height="101" style="height:101px;width:216px;">Computer program for measuring staining intensity</td>
			<td style="width:71px;">Image J</td>
			<td style="width:119px;">https://imagej.nih.gov/ij/ 
			Program and information available on-line</td>
			<td style="width:361px;">Any appropriate program can be used. See https://theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html for additional detail&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell Culture trypsin solution</td>
			<td style="width:71px;">Sigma</td>
			<td>T4174</td>
			<td style="width:361px;">purchased as a 10X solution</td>
		</tr>
	</tbody>
</table>

using a gfp-tagged tmem184a construct for confirmation of heparin receptor identity

When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies against specific peptides within the predicted sequence allow some localization experiments. However, other possible interactions with the antibodies often cannot be excluded. This situation provided an opportunity to develop a set of assays dependent on the protein sequence. Specifically, a construct containing the gene sequence coupled to the GFP coding sequence at the C-terminal end of the protein was obtained and employed for these purposes. Experiments to characterize localization, ligand affinity, and gain of function were originally designed and carried out to confirm the identification of TMEM184A as a heparin receptor1. In addition, the construct can be employed for studies addressing membrane topology questions and detailed protein-ligand interactions. The present report presents a range of experimental protocols based on the GFP-TMEM184A construct expressed in vascular cells that could easily be adapted for other novel proteins.

While, in theory, transfection of any DNA construct into cells could be accomplished with lipophilic transfection reagents, previous reports indicate more effective transfection of GFP constructs into endothelial cells using electroporation12. The protocol provided here typically achieved GFP-construct expression in greater than 80% of the primary-derived endothelial cells and smooth muscle cells used. Design of the construct employed used a commercially available ...

Watch this Scientific Journal Video about Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity at JoVE.com

Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity

A construct encoding TMEM184A with a GFP tag at the carboxy-terminus designed for eukaryotic expression, was employed in assays designed to confirm the identification of TMEM184A as a heparin receptor in vascular cells.

When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies ...

The overall goal of using a GFP-tagged construct is to confirm the identity of a previously unknown protein using the GFP as a handle. This method can help with the functional identification of orphan genes when a specific enzyme activity is not available for confirmation. The main advantage of this technique is that the GPF-tag allows the same construct to be used for several types of experiments.Generally, individuals who are new to this method may have trouble, because the transfection does not always result in the strong-construct expression, and the cells do not always revive. We first had the idea for this method, when we began to recognize the difficulties of identifying a protein using only antibodies created against it. Begin by resuspending the cells of interest in one milliliter of HEPES-buffered saline electroporation buffer and immediately place the cells on ice.Add a sufficient volume of GFP-TMEM184A-tagged plasma to the cells to achieve a final concentration of 20 micrograms per milliliter of DNA, and transfer approximately 0.4 milliliters of cells into a pre-chilled electroporation cuvette. Electroporate the cells. Then see the transfected cells onto tissue culture dishes, and place the dishes in a cell culture incubator for 24 to 72 hours.The transfection step is the most critical step of the procedure, because most of the remaining experimental work cannot be effectively completed if the cells are not viable, and do not express significant levels of the GFP-tagged construct. At the appropriate experimental time point, add 100 micrograms per milliliter of Rhodamine Heparin to the GFP-plasmid-expressing cell culture medium, and incubate the cells, protected from light, until the desired level of staining is reached as observed by fluorescence microscopy. Next, rinse the cells with PBS and a few seconds of gentle shaking, protected from light, followed by fixation with 4%paraformaldehyde for 15 minutes at room tempurature with gentle shaking.To assess the GFP-tagged Rhodamine Heparin co-localization, use a confocal microscope with the appropriate GFP and Rhodamine excitation and diminished lasers to image at least 50 cells from each separate labeling experiment. Examine the images in the appropriate image-analysis program to determine the relative Rhodamine finding and uptake in each cell, using a free-hand tool to circle the cells within each fluorescent image. Use the measure tool to determine the intensity within each circled space, then export the fluorescence-intensity data to a spreadsheet to calculate the area multiplied by the intensity, subtracting the background for the same amount of area for each cell.The fluorescence per cell can then be averaged for all of the cells, to obtain statistically significant data. To facilitate the fluorescence resonance energy transfer from GFP TMEM184A to Rhodamine Heparin, first mount fixed GFP TMEM184A transfected Rhodamine Heparin-labeled cells onto glass microscope slides. Excite the cells at 405 nanometers for imaging the Rhodamine emission, followed by excitation of 488 nanometers to image the GFP emission.Then excite the cells at 561 nanometers for a second Rhodamine emission imaging. For live-cell imaging of the Rhodamine Heparin uptake, seed a 100-milliliter confluent dish culture of the GFP TMEM184A-transfected cells into 635 millimeter live-cell imaging dishes to obtain cells near confluence. After 48 hours, replace the supernatant with culture medium without Phenol red, and transfer a dish of cells to a confocal microscope with a warming stage set to 37 degrees Celsius.Focus the microscope, and add 100 micrograms per milliliter of Rhodamine Heparin to the cells with gentle mixing. Then immediately begin recording the live images. If no Heparin uptake occurs within the field of view, move the dish slightly to identify the cells with at least one green vesicle, containing the Rhodamine label.For Heparin-binding assessment in vitro, rinse one black ativan-coated 96 well plate and one black non-coated 96 well plate with 200 microliters of 0.2%CHAPS PBS pro-well for three five-minute washes with shaking. After the last wash, add 100 microliters of 60 picomoles per well of biotinylated anti-GFP in triplicate to the ativan-coated plate for each GFP finding group. Next, add buffer only to the control wells and seal all of the wells with a plate cover for a two-hour incubation at room tempurature with shaking.At the end of the incubation, wash all of the wells three times in CHAPS PBS, as just demonstrated, and add 100 microliters of five nanomoles per well of GFP TMEM184A, or GFP alone, to the appropriate wells in the ativan-coated plate. After a one-hour incubation at room tempurature with shaking, wash all of the wells with fresh CHAPS PBS, and add 100 microliters of each experimental fluorescein-Heparin concentration, to the appropriate test wells in triplicate, to the ativan-coated plate. Add the concentration standards to both the ativan-coated and non-coated plates, and incubate both plates for 10 minutes at room tempurature, with shaking, protected from light.Now, use a plate reader to record the initial Heparin-fluorescence emission in the control wells of both plates, and the initial total fluorescence emission from the wells with the immobilized GFP or GFP TMEM184A in the ativan-coated plate. Then transfer the unbound fluorescent Heparin from the wells with the immobilized GFP TMEM184A or GFP into the corresponding wells in the non-coated black plate. After immediately reading the fluorescence emission on the non-coated black plate, add 100 microliters of fresh CHAPS PBS back into wells from which the fluorescein-Heparin was removed, and read the fluorescence emissions for these wells to obtain the fluorescence emission from the removed, unbound samples in the non-coated plate.When all of the unbound readings have been obtained, use the total Heparin emission values to confirm the correct levels of Heparin were added to each well, by subtracting the average background readings from the values. Then create a plot of the emission versus the total fluorescein-Heparin added, to determine the scale of the emission versus the amount of Heparin, and plot the corrected bound Heparin, versus the added Heparin for the triplicates from both of the GFP and GFP TMEM184A wells. The length of time after transfection impacts the distribution of the GFP TMEM184A, which appears to accumulate over at least 72 hours, with more GFP apparent in the paranuclear region over time.The staining pattern of GFP in this representative vascular cell culture, appears to be most similar to that of an antibody raised against a C-terminal peptide, a logical outcome, given that the GFP is at the C-terminus of the protein. The use of a fluorescein-Rhodamine-Heparin ligand can also be used for the real-time evaluation of ligand receptor co-localization over time. Heparin uptake is increased in GFP TMEM184A construct transfected stable knockdown cells, resulting in cells that can regain the ability to internalize Rhodamine Heparin at or above the level of wild-type cells.Using the demonstrated plasmid-encoded GFP tact system, the anti-GFP antibody isolated GFP TMEM184A construct demonstrates a specific satriple Heparin binding, while the control GFP does not bind any Heparin. Simple immunofluorescence staining with antibodies again GFP can also provide information about the GFP location, based on the antibody access in non-permeabilized cells, providing preliminary evidence that the GFP expressed at the plasma membrane is extra-cellular. Once mastered, this technique can result in excellent GFP-construct expression, after 24 to 48 hours of culture.While attempting the live imaging protocol, it is important to remember that some cells might not exhibit the co-localized uptake, so it is often necessary to examine other cells. Binding assays with GFP ligands can be complicated by dimer or higher-order aggregates of the GFP protein, particularly if they are in detergent micelles. Overall, the expression of GFP-tagged proteins can facilitate a wide variety of experiments to confirm the identity of a previously-unknown protein.

using-gfp-tagged-tmem184a-construct-for-confirmation-heparin-receptor

Research

JoVE Journal

Genetics

7.9K Görüntüleme.  Lehigh University. The overall goal of using a GFP-tagged construct is to confirm the identity of a previously unknown protein using the GFP as a handle. This method can help with the functional identification of orphan genes when a specific enzyme activity is not available for confirmation. The main advantage of this technique is that the GPF-tag allows the same construct to be used for several types of experiments.Generally, individuals who are new to this method may have trouble, because the transfect...