We are very grateful to Dr. Brigitte Sturm for her support with the ECLIA instrument.

Approval for skin biopsy procurement for research purposes was obtained from the Human Research Ethics Committee of the Children&#8217;s Hospital at Westmead, Australia. Consent for animal experiments was obtained from the Austrian Federal Ministry of Science, Research and Economy, which were performed in accordance with local animal welfare regulations (GZ: 66.009/0218-II/3b/2015).
NOTE: The principle of the ECLIA system is depicted in Figure 1.
<p class="jov...

<ol>
	<li>Debad, J. D., Glezer, E. N., Wohlstadter, J., Sigal, G. B., Leland, J. K., Bard, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+biological+applications+of+ECL.">Clinical and biological applications of ECL.</a> Electrogenerated Chemiluminescence. , 359-383 (2004).</li><li>Yuzaburo, N., Michinori, U., Osamu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Sensitive+Electrochemiluminescence+Immunoassay+Using+the+Ruthenium+Chelate-Labeled+Antibody+Bound+on+the+Magnetic+Micro+Beads.">Highly Sensitive Electrochemiluminescence Immunoassay Using the Ruthenium Chelate-Labeled Antibody Bound on the Magnetic Micro Beads.</a> Analytical Sciences. 15 (11), 1087-1093 (1999).</li><li>Liu, W., Hu, Y., Yang, Y., Hu, T., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+immunoassays+for+quantification+of+hepatitis+B+surface+antigen+in+Chinese+patients+with+concomitant+hepatitis+B+surface+antigen+and+hepatitis+B+surface+antibodies.">Comparison of two immunoassays for quantification of hepatitis B surface antigen in Chinese patients with concomitant hepatitis B surface antigen and hepatitis B surface antibodies.</a> Archives of Virology. 160 (1), 191-198 (2015).</li><li>Shah, R. R., Bird, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MeCP2+mutations:+progress+towards+understanding+and+treating+Rett+syndrome.">MeCP2 mutations: progress towards understanding and treating Rett syndrome.</a> Genome Medicine. 9 (1), 17 (2017).</li><li>Chahrour, 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=MeCP2,+a+key+contributor+to+neurological+disease,+activates+and+represses+transcription.">MeCP2, a key contributor to neurological disease, activates and represses transcription.</a> Science. 320 (5880), 1224-1229 (2008).</li><li>Lyst, M. J., Bird, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rett+syndrome:+a+complex+disorder+with+simple+roots.">Rett syndrome: a complex disorder with simple roots.</a> Nature Reviews Genetics. 16 (5), 261-275 (2015).</li><li>Katz, D. 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=Rett+Syndrome:+Crossing+the+Threshold+to+Clinical+Translation.">Rett Syndrome: Crossing the Threshold to Clinical Translation.</a> Trends in Neurosciences. 39 (2), 100-113 (2016).</li><li>Steinkellner, H., 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=An+electrochemiluminescence+based+assay+for+quantitative+detection+of+endogenous+and+exogenously+applied+MeCP2+protein+variants.">An electrochemiluminescence based assay for quantitative detection of endogenous and exogenously applied MeCP2 protein variants.</a> Scientific Reports. 9 (1), 7929 (2019).</li><li>Laccone, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Synthetic MeCP2 sequence for protein substitution therapy. , (2007).</li><li>Koutsokeras, A., Kabouridis, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretion+and+uptake+of+TAT-fusion+proteins+produced+by+engineered+mammalian+cells.">Secretion and uptake of TAT-fusion proteins produced by engineered mammalian cells.</a> Biochimica et Biophysica Acta (BBA) - General Subjects. 1790 (2), 147-153 (2009).</li><li>Suzuki, K., Bose, P., Leong-Quong, R. Y., Fujita, D. J., Riabowol, K. R. E. A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REAP:+A+two+minute+cell+fractionation+method.">REAP: A two minute cell fractionation method.</a> BMC Research Notes. 3, 294 (2010).</li><li>Xia, H., Mao, Q., Davidson, B. 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+HIV+Tat+protein+transduction+domain+improves+the+biodistribution+of+beta-glucuronidase+expressed+from+recombinant+viral+vectors.">The HIV Tat protein transduction domain improves the biodistribution of beta-glucuronidase expressed from recombinant viral vectors.</a> Nature Biotechnology. 19 (7), 640-644 (2001).</li><li>Schwarze, S. R., Ho, A., Vocero-Akbani, A., Dowdy, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+protein+transduction:+delivery+of+a+biologically+active+protein+into+the+mouse.">In vivo protein transduction: delivery of a biologically active protein into the mouse.</a> Science. 285 (5433), 1569-1572 (1999).</li><li>Nagahara, H., 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=Transduction+of+full-length+TAT+fusion+proteins+into+mammalian+cells:+TAT-p27Kip1+induces+cell+migration.">Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration.</a> Nature Medicine. 4 (12), 1449-1452 (1998).</li><li>Trazzi, 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=CDKL5+protein+substitution+therapy+rescues+neurological+phenotypes+of+a+mouse+model+of+CDKL5+disorder.">CDKL5 protein substitution therapy rescues neurological phenotypes of a mouse model of CDKL5 disorder.</a> Human Molecular Genetics. 27 (9), 1572-1592 (2018).</li><li>Van Esch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MECP2+Duplication+Syndrome.">MECP2 Duplication Syndrome.</a> Molecular Syndromology. 2 (3-5), 128-136 (2012).</li><li>Collins, A. 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=Mild+overexpression+of+MeCP2+causes+a+progressive+neurological+disorder+in+mice.">Mild overexpression of MeCP2 causes a progressive neurological disorder in mice.</a> Human Molecular Genetics. 13 (21), 2679-2689 (2004).</li><li>Luikenhuis, S., Giacometti, E., Beard, C. F., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+MeCP2+in+postmitotic+neurons+rescues+Rett+syndrome+in+mice.">Expression of MeCP2 in postmitotic neurons rescues Rett syndrome in mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (16), 6033-6038 (2004).</li></ol>

The authors have nothing to disclose.

To measure endogenous MeCP2, recombinant MeCP2 and TAT-MeCP2 levels, a 96-well plate ECLIA was developed. It has been shown that loss of MeCP2 protein function leads to RTT syndrome6, for which treatment is currently limited to symptom management and physical therapy. One promising treatment avenue is the so-called protein replacement therapy, where MeCP2 levels can be titrated up to their needed concentration12,13,<sup class="x...

The electrochemiluminescence immunoassay (ECLIA) is based on a process that utilizes labels designed to emit luminescence when electrochemically stimulated. It is a broadly applicable technique for the quantitative detection of biological analytes in basic industry and academic research, food industry as well as in clinical diagnostics1. Commonly, a disposable 96-well plate with carbon ink electrodes is used. These electrodes act as a solid-phase carrier for the immunoassay. A secondary antibody is conjugated to an electrochemiluminescent label and when electricity is applied to the system, light emission of the chemical label is triggered. An ultra-low noise charge-coupled device (CCD) records the light intensity which is directly proportional to the antigen bound to the capture antibody resulting in the quantification of the targeted analyte of the sample2. Compared to the enzyme-linked immunosorbent assay (ELISA), ECLIA is considered to be advantageous as it offers higher sensitivity and reproducibility as well as better automation and consistency3.
Here we analyzed the methyl-CpG binding protein 2 (MeCP2) levels in samples of human and murine origin as well as different variants of the recombinant protein using the newly developed ECLIA system. MecP2 is an X-linked nucleic acid-binding protein known to interact with methylated DNA sequences. This protein has been implicated in the regulation of gene expression4,5. Loss-of-function mutations in the gene which encodes this protein are the main culprits causing Rett syndrome (RTT), a severe neurodevelopmental disorder6. Another MeCP2-related disorder, MECP2 duplication syndrome, also leads to neurological symptoms that can overlap with those of RTT7. Notably, females are mostly affected by RTT while males are mostly afflicted by MECP2 duplication syndrome6,7.
These disorders are associated with insufficient or excess MeCP2 levels respectively in the central nervous system (CNS). Hence, treatment options for RTT that involve increasing MeCP2 levels in the CNS would need to avoid the detrimental effects associated with excess of MeCP27. Due to this fact, a highly sensitive and accurate quantification of MeCP2 protein levels, as provided by the ECLIA system, is crucial for the advancement of RTT as well as MECP2 duplication syndrome research. The precise measurements of endogenous and exogenous MeCP2 levels from human cell lines and mouse tissue samples as well as a recombinant protein consisting of human MeCP2 isoform B (also known as isoform e1), and a minimal N-terminal HIV-TAT transduction domain (TAT-MeCP2) that has the potential to cross the blood-brain-barrier8,9 are presented in this work.

<table>
	<tbody>
		<tr>
			<td>1,4-Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>D9779</td>
			<td>Hypotonic lysis reagent, Extraction Buffer</td>
		</tr>
		<tr>
			<td>Bio-Rad Protein Assay Dye Reagent Concentrate</td>
			<td>Bio-Rad Laboratories Inc.</td>
			<td>500-0006</td>
			<td>Sample preperation</td>
		</tr>
		<tr>
			<td>Detection AB SULFO-TAG labeled anti-rabbit</td>
			<td>Meso Scale Diagnostics</td>
			<td>R32AB-1</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Discovery workbench 4.0</td>
			<td>Meso Scale Discovery</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>DMEM (1X)</td>
			<td>gibco by Life Technologies</td>
			<td>41966-029</td>
			<td>Sample preperation</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s PBS (sterile)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537-500ML</td>
			<td>Sample preperation, Washing solution, Coating solution</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>EDS</td>
			<td>Extraction Buffer</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F9665</td>
			<td>Sample preperation</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G2025</td>
			<td>Extraction Buffer</td>
		</tr>
		<tr>
			<td>Gold Read Buffer T (1x) with surfactant</td>
			<td>Meso Scale Diagnostics</td>
			<td>R92TG</td>
			<td>MeCP2 ECLIA protocol</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Hypotonic lysis reagent, Extraction Buffer</td>
		</tr>
		<tr>
			<td>KIMBLE Dounce tissue grinder set</td>
			<td>Sigma-Aldrich</td>
			<td>D8938</td>
			<td>Sample preperation</td>
		</tr>
		<tr>
			<td>Laboratory Shaker, rocking motion (low speed)</td>
			<td>GFL</td>
			<td>3014</td>
			<td>MeCP2 ECLIA protocol</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate (MgCl*6H20)</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td>Hypotonic lysis reagent, Extraction Buffer</td>
		</tr>
		<tr>
			<td>MeCP2 (Human) Recombinant Protein (P01)</td>
			<td>Abnova Corporation</td>
			<td>H00004204-P01</td>
			<td>Cell treatment</td>
		</tr>
		<tr>
			<td>Microseal B seal</td>
			<td>Bio-Rad Laboratories Inc.</td>
			<td>MSB1001</td>
			<td>for plate sealing</td>
		</tr>
		<tr>
			<td>Monoclonal Anti-MeCP2, produced in mouse, clone Mec-168, purified immunoglobulin</td>
			<td>Sigma-Aldrich</td>
			<td>M6818-100UL; RRID:AB_262075</td>
			<td>Antibody, Coating solution</td>
		</tr>
		<tr>
			<td>MSD Blocker A</td>
			<td>Meso Scale Diagnostics</td>
			<td>R93BA-4</td>
			<td>Blocker</td>
		</tr>
		<tr>
			<td>MSD SECTOR Imager 2400</td>
			<td>Meso Scale Diagnostics</td>
			<td>I30AA-0</td>
			<td>MeCP2 ECLIA protocol</td>
		</tr>
		<tr>
			<td>Multi-Array 96-well Plate</td>
			<td>Meso Scale Diagnostics</td>
			<td>L15XB-3/L11BX-3</td>
			<td>MeCP2 ECLIA protocol</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>gibco by Life Technologies</td>
			<td>15140122</td>
			<td>Sample preperation</td>
		</tr>
		<tr>
			<td>Polyclonal Anti-MeCP2, produced in rabbit</td>
			<td>Eurogentec S.A.</td>
			<td>custom-designed</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Merck KGaA</td>
			<td>1049361000</td>
			<td>Hypotonic lysis reagent</td>
		</tr>
		<tr>
			<td>Primary AB Mouse, anti-MeCP2 (1B11)</td>
			<td>Sigma-Aldrich</td>
			<td>SAB1404063; RRID:AB_10737296</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Primary AB Mouse, anti-MeCP2 (4B6)</td>
			<td>Sigma-Aldrich</td>
			<td>WH0004204M1; RRID:AB_1842411</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Primary AB Mouse, anti-MeCP2 (Mec-168)</td>
			<td>Sigma-Aldrich</td>
			<td>M6818; RRID:AB_262075</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Primary AB Mouse, anti-MeCP2 (Men-8)</td>
			<td>Sigma-Aldrich</td>
			<td>M7443; RRID:AB_477235</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Primary AB Rabbit, anti-MeCP2 (D4F3)</td>
			<td>Cell Signaling Technology</td>
			<td>3456S; RRID:AB_2143849</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail (100X)</td>
			<td>Sigma-Aldrich</td>
			<td>8340</td>
			<td>Hypotonic lysis reagent, Extraction Buffer</td>
		</tr>
		<tr>
			<td>Secondary AB, Rabbit, anti-MeCP2</td>
			<td>Eurogentec S.A.</td>
			<td>custom</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Secondary AB, Rabbit, anti-MeCP2</td>
			<td>Merck</td>
			<td>07-013</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td>Extraction Buffer</td>
		</tr>
		<tr>
			<td>SULFO-TAG Labeled Anti-Rabbit Antibody (goat)</td>
			<td>Meso Scale Diagnostics</td>
			<td>W0015528S</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>TAT-MeCP2 fusion protein</td>
			<td>in-house production</td>
			<td></td>
			<td>Cell treatment</td>
		</tr>
		<tr>
			<td>Trypsin EDTA 0.25% (1X)</td>
			<td>gibco by Life Technologies</td>
			<td>25200-056</td>
			<td>Cell treatment</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td>Washing solution</td>
		</tr>
	</tbody>
</table>

an electrochemiluminescence-based assay for mecp2 protein variants

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.

The principle of the ECLIA system is described in Figure 1. Standard curves for two MeCP2 variants are shown in Figure 2. Accurate quantification was possible over a wide range of concentrations (1&#8722;1,800 ng/mL). In Figure 3, MeCP2 levels of lysates derived from mouse brain and HDFs were analyzed. MeCP2 expression in brain nuclear lysates from heterozygous, wildtype and knockout mice were compared in Figure...

Watch this Scientific Journal Video about An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants at JoVE.com

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

The electrochemiluminescence immunoassay (ECLIA) is a novel approach for quantitative detection of endogenous and exogenously applied MeCP2 protein variants, which produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error over a wide working range. Here, the protocol for the MeCP2-ECLIA in a 96-well format is described.

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, ...

The ECLIA is a simple yet efficient method to quantify cellular levels of MeCP2. The ECLIA is fast, more sensitive, and easier to handle compared to other quantification methods such as the Western blot and the ELISA. To prepare the washing solution, which is 0.5%Tween 20 in PBS, add 500 microliters of Tween 20 to one liter of PBS and mix vigorously.Prepare a blocking solution of 3%Blocker A and PBS and stir gently. Sterilize the blocking solution by filtration. To prepare the assay diluent solution, 1%Blocker A, and PBS, add five milliliters of blocking solution to 10 milliliters of PBS.Next thaw the monoclonal mouse anti-MeCP2 antibody on ice. Mix 0.67 microliters of the antibody with four milliliters of PBS, then vortex the solution. To solution coat the 96-well high bind plate, carefully dispense 25 microliters of coating solution into the bottom corner of each well, using a multichannel pipetter.Dab the 96-well plate gently on each side to ensure that the coating solution covers the bottom of each well. Seal the plate with adhesive foil, and incubate overnight at four degrees Celsius. Retrieve the 96-well plate from the refrigerator and remove the foil.Remove the antibody coating solution in the wells by flicking it into a waste container. Then tap the plate on a paper towel to remove any remaining solution. Next, add 125 microliters of blocking solution to each well.Then seal the plate again with adhesive foil. Place the plate on an orbital microplate shaker, set at 800 RPM, and incubate it for 90 minutes at room temperature. Thaw on ice one vial of MeCP2 or Tat-MeCP2 protein stock solution, mouse brain lysates, and HDF lysates.Dilute the protein stock solution in clean tubes as described in table two of the manuscript. Next dilute the lysate samples. For the mouse brain lysates, use one to 20 micrograms per 25 microliters of lysis buffer.For the HDF lysate, add 0.25 to one microgram per 25 microliters of lysis buffer. After the 96-well plate has been incubating for 90 minutes, remove the blocking solution from the wells by flicking it into a waste container. Then tap the plate on a paper towel to remove any remaining solution.Then wash the plate three times with 150 microliters of washing solution, by adding the washing solution and immediately removing it. Add 25 microliters of standards and samples to the wells of the 96-well plate. Seal the plate and incubate it for another four hours at room temperature with constant 800 RPM shaking.Thaw the unlabeled detection antibody, polyclonal rabbit anti-MeCP2, on ice. Using assay diluent solution, dilute the antibody at a ratio of one to 6, 000. When the 96-well plate is finished incubating on the shaker, remove the standards and samples by flicking the plate into a waste container.Then tap the plate on a paper towel to remove any remaining solution. Wash the plate three times with 150 microliters of washing solution by adding the washing solution and immediately removing it. Add 25 microliters of unlabeled detection antibody solution to each well with the multichannel pipetter.Seal the plate and incubate it for one hour at room temperature with constant shaking at 800 RPM. Obtain the specific secondary antibody from the refrigerator and place it one ice. Dilute the secondary body in assay diluent solution and mix gently.To remove the free unlabeled detection antibody from the 96-well plate, flick in into the waste container and then tap the plate on a paper towel. After washing the plate three times as perviously described, add 25 microliters of secondary antibody to each well with the multichannel pipetter. Seal the plate and incubate it for one hour at room temperature with constant shaking at 800 RPM.Remove the free secondary antibody by flicking in into the waste container and tapping the plate on a paper towel and then wash the plate three times, as previously described. To each well of the plate, add 150 microliters of 1X Tris base Gold Read Buffer with surfactant, containing tripropylamine as a coreactant for light generation. To avoid producing air bubbles, use reverse pipetting techniques.Place the 96-well plate on the microplate platform with electrochemiluminescence detection system. Using the built in CCD camera, immediately begin capturing data. Record signal counts which correspond to relative light units and are directly proportional to the intensity of light.MeCP2 standard curves were generated from human MeCP2 in multiple measurements. The ECLIA can accurately quantify recombinant human MeCP2 over a range from one nanogram per milliliter to 1800 nanograms per milliliter. Using ECLIA, MeCP2 protein levels were measured in brain nuclear lysates from heterozygous and female wild-type mice and from one MeCP2 knockout mouse.ECLIA was conducted on cell lysates from a healthy control and from the c. 806delG cell line used as a model for Rett syndrome. No MeCP2 protein was detected in the mutant cell lines by ECLIA or by immunofluorescence.ECLIA was also used to investigate the uptake of Tat-MeCP2 by the MeCP2 deficient cell line overtime. ECLIA inter and intra assay precision was determined over three consecutive days. For future protein replacement therapy, MeCP2 delivery can be repeatedly optimized following protein level assessment by the ECLIA.

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JoVE Journal

Biochemistry

26.4K visualizações.  Medical University of Vienna (MUV). O ECLIA é um método simples, mas eficiente, para quantificar os níveis celulares de MeCP2. O ECLIA é rápido, mais sensível e mais fácil de manusear em comparação com outros métodos de quantificação, como a mancha ocidental e a ELISA. Para preparar a solução de lavagem, que é 0,5%Tween 20 em PBS, adicione 500 microliters de Tween 20 a um litro de PBS e misture vigorosamente.Prepare uma solução de bloqueio de 3% Bloqueador A e PBS e mexa suavemente. Esterilize a soluçã...