Name: highly sensitive and quantitative detection of proteins and their isoforms by capillary isoelectric focusing method
Uploaded: 2018-09-19T12:30:00
Description: Watch this Scientific Journal Video about Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method at JoVE.com

The author thanks Prof. Lena Claesson-Welsh, Uppsala University, Sweden for her support for developing this project. Additionally, the author thanks Ross Smith and Lena Claesson-Welsh, Uppsala University for their critical reading and suggestions to improve the manuscript.

1. Cell Culture, Stimulation, and Lysis
Note: This method can be used with many cell types. To illustrate the method, an example using human umbilical vein endothelial cells (HUVECs) is described.
<ol>
	<li>Culture HUVECs on gelatin-coated, 10 cm Petri dishes in endothelial cell basal medium with appropriate supplementation (see Table of Materials), and also containing 5% FCS, epidermal growth factor (5 ng/mL), vascular endothelial growth factor (VEGF: 0.5 ng/mL), basic FGF ...

<ol>
	<li>O'Neill, R. 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=Isoelectric+focusing+technology+quantifies+protein+signaling+in+25+cells.">Isoelectric focusing technology quantifies protein signaling in 25 cells.</a> Proc Natl Acad Sci U S A. 103 (44), 16153-16158 (2006).</li><li>Aspinall-O'Dea, 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=Antibody-based+detection+of+protein+phosphorylation+status+to+track+the+efficacy+of+novel+therapies+using+nanogram+protein+quantities+from+stem+cells+and+cell+lines.">Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines.</a> Nat Protoc. 10 (1), 149-168 (2015).</li><li>Fan, 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=Nanofluidic+proteomic+assay+for+serial+analysis+of+oncoprotein+activation+in+clinical+specimens.">Nanofluidic proteomic assay for serial analysis of oncoprotein activation in clinical specimens.</a> Nat Med. 15 (5), 566-571 (2009).</li><li>Padhan, N., 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=High+sensitivity+isoelectric+focusing+to+establish+a+signaling+biomarker+for+the+diagnosis+of+human+colorectal+cancer.">High sensitivity isoelectric focusing to establish a signaling biomarker for the diagnosis of human colorectal cancer.</a> BMC Cancer. 16 (1), 683 (2016).</li><li>Sun, 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=VEGFR2+induces+c-Src+signaling+and+vascular+permeability+in+vivo+via+the+adaptor+protein+TSAd.">VEGFR2 induces c-Src signaling and vascular permeability in vivo via the adaptor protein TSAd.</a> J Exp Med. 209 (7), 1363-1377 (2012).</li><li>Iacovides, D. 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=Identification+and+quantification+of+AKT+isoforms+and+phosphoforms+in+breast+cancer+using+a+novel+nanofluidic+immunoassay.">Identification and quantification of AKT isoforms and phosphoforms in breast cancer using a novel nanofluidic immunoassay.</a> Mol Cell Proteomics. 12 (11), 3210-3220 (2013).</li><li>Price, F. 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=Inhibition+of+JAK-STAT+signaling+stimulates+adult+satellite+cell+function.">Inhibition of JAK-STAT signaling stimulates adult satellite cell function.</a> Nat Med. 20 (10), 1174-1181 (2014).</li><li>Unger, F. T., 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=Nanoproteomic+analysis+of+ischemia-dependent+changes+in+signaling+protein+phosphorylation+in+colorectal+normal+and+cancer+tissue.">Nanoproteomic analysis of ischemia-dependent changes in signaling protein phosphorylation in colorectal normal and cancer tissue.</a> J Transl Med. 14, 6 (2016).</li><li>Urasaki, Y., Pizzorno, G., Le, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+uridine+administration+induces+fatty+liver+and+pre-diabetic+conditions+in+mice.">Chronic uridine administration induces fatty liver and pre-diabetic conditions in mice.</a> PLoS One. 11 (1), e0146994 (2016).</li><li>Schmidt, 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=Case-specific+potentiation+of+glioblastoma+drugs+by+pterostilbene.">Case-specific potentiation of glioblastoma drugs by pterostilbene.</a> Oncotarget. 7 (45), 73200-73215 (2016).</li></ol>

Sensitivity and resolution of proteins are crucial for proteomic research on biological samples. There is great value in being able to detect proteins which are present in minute quantities in cells. cIEF can offer improved sensitivity and resolution for the detection of proteins and their isoforms4.
This technology has been successfully used in many proteomic research reports5,6,7</...

Capillary isoelectric focusing (cIEF) is an automated, capillary-based immunoassay that resolves proteins on the basis of their charge1,2,3. It is highly reproducible and capable of resolving proteins and their post-translationally modified isoforms rapidly and quantitatively. It presents an alternative to conventional methods such as western blotting. While western blotting is very good for confirming the presence of abundant proteins in readily accessible samples; variability, time consumption, and accurate quantitation all present challenges, in particular when examining biological tissue samples. Indeed, variability is an inherent problem in western blotting, as there are numerous steps involved, such as loading and running of SDS-PAGE gels, transfer of proteins onto membrane, incubation with various reagents (e.g., primary and secondary antibodies, ECL), and development onto X-ray film4. Presently, the western blotting technique is improving with the implementation of digital recording of chemiluminescent signals (digital westerns). Recently, an automated western blotting system has been developed, namely the capillary western, which is a more hands-free and gel-free system. The entire assay is automated following the loading of a sample plate (samples with all necessary reagents) into the system3,4. The instrument will perform all the steps such as protein separation, immobilization of proteins onto capillary wall, antibody incubations, washes between different steps, and development and quantification of the chemiluminescent signals. Thus, the cIEF procedure presented here provides higher resolution and sensitivity.
This method is sensitive, as signals can be generated and quantified from picograms of proteins1. The high sensitivity with excellent reproducibility makes this technology very useful for the analysis of clinical samples. It can detect as well as distinguish post translational modification (e.g., different phosphorylated protein isoforms) of proteins. This technology has been successfully used to dissect different signaling pathways4,5 in clinical studies aiming to develop new therapeutics in cancer3, and it has great potential for protein biomarker and drug discovery.

<table border="0" cellpadding="0" cellspacing="0" width="920">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">NanoPro 1000</td>
			<td style="width:107px;">ProteinSimple</td>
			<td style="width:301px;"></td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Premix G2, pH 5&#8211;8 (nested) separation gradient, pH 2&#8211;4 plug</td>
			<td>ProteinSimple</td>
			<td>040-972</td>
			<td>Ampholyte premix</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMSO inhibitor mix</td>
			<td>ProteinSimple</td>
			<td>040-510</td>
			<td>Phosphatase inhibitors; for cIEF 1:50 dilution used</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Aqueous Inhibitor Mix</td>
			<td>ProteinSimple</td>
			<td>040-482</td>
			<td>Protease inhibitor; for cIEF 1:25 dilution used</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">pI standard ladder 3</td>
			<td>ProteinSimple</td>
			<td>040-646</td>
			<td>For cIEF 1:60 dilution used</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sample diluent</td>
			<td>ProteinSimple</td>
			<td>040-649</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Antibody diluent&#160;</td>
			<td>ProteinSimple</td>
			<td>040-309</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Wash concentrate</td>
			<td>ProteinSimple</td>
			<td>041-108</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anolyte Refill</td>
			<td>ProteinSimple</td>
			<td>040-337</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Catholyte Refill</td>
			<td>ProteinSimple</td>
			<td>040-338</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Peroxide XDR</td>
			<td>ProteinSimple</td>
			<td>041-084</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Luminol</td>
			<td>ProteinSimple</td>
			<td>040-652</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bicine/CHAPS Lysis Buffer</td>
			<td>ProteinSimple</td>
			<td>040-764</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Capillaries-Charge Separation (1 pack) for&#160;</td>
			<td>ProteinSimple</td>
			<td>CBS700</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Assay Plate/Lid Kit</td>
			<td>ProteinSimple</td>
			<td>040-663</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Peroxidase-AffiniPure Donkey Anti-Rabbit IgG (H+L)&#160;</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>711-035-152</td>
			<td>For cIEF used (1: 300)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Peroxidase-AffiniPure Donkey Anti-Mouse IgG (H+L)&#160;</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>715-035-150</td>
			<td>For cIEF used (1: 300)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Phospho-Erk 1/2 Primary Antibody</td>
			<td>Cell Signaling</td>
			<td>9101</td>
			<td>For cIEF used (1: 50)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pan Erk Primary Antibody</td>
			<td>Cell Signaling</td>
			<td>9102</td>
			<td>For cIEF used (1: 100)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Compass software</td>
			<td>ProteinSimple</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HUVEC</td>
			<td>ATCC</td>
			<td>PCS-100-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MV2 (EBM-2)</td>
			<td>PromoCell</td>
			<td>&#160;C-22221</td>
			<td>Endothelia cell basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endothelial Cell Growth Medium MV2 SupplementPack</td>
			<td>PromoCell</td>
			<td>C-39221</td>
			<td>Supplementation to MV2 above</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VEGFA</td>
			<td>PeproTech</td>
			<td>100-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Long R3 IGF</td>
			<td>Sigma-Aldrich</td>
			<td>85580C/I1146</td>
			<td>Insulin-like growth factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bioruptor (Sonicator)</td>
			<td>Diagenode</td>
			<td>B01020001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BCA Protein Assay kit&#160;</td>
			<td>Thermo Fischer Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE 4-12% Bis-Tris Protein Gels</td>
			<td>Thermo Fischer Scientific</td>
			<td>NP0322BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAG MOPS SDS Running Buffer (20X)</td>
			<td>Thermo Fischer Scientific</td>
			<td>NP0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE Transfer Buffer (20X)</td>
			<td>Thermo Fischer Scientific</td>
			<td>NP0006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immobilon PVDF membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microfuge tube vortexer</td>
			<td></td>
			<td></td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Centrifuge</td>
			<td style="width:107px;"></td>
			<td style="width:301px;"></td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Microtiter plate adapter for centrifuge</td>
			<td style="width:107px;"></td>
			<td style="width:301px;"></td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Pipettors&#160;</td>
			<td style="width:107px;"></td>
			<td style="width:301px;"></td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Tips</td>
			<td style="width:107px;"></td>
			<td style="width:301px;"></td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Ice</td>
			<td style="width:107px;"></td>
			<td style="width:301px;"></td>
			<td style="width:147px;"></td>
		</tr>
	</tbody>
</table>

highly sensitive and quantitative detection of proteins and their isoforms by capillary isoelectric focusing method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Design of a new assay: An assay plate layout is shown in Figure 1A. Maximally, 96 wells can be used from the 384-well plate in blocks of 12 wells for each condition (antibody). Each block of 12 wells can start either from A1-A12 or A13-A24. Color coded rows allow one to distinguish samples or reagents from one another. In the assay template (Figure 1B), relevant information for that particular assay can be stored...

Watch this Scientific Journal Video about Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method at JoVE.com

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

This method can help answer key questions in the biomedical research field, such as biomarker discovery, drug discovery, diagnostics, and screening of drugs or antibodies. The main advantage of this technique is that it is high-throughput and can detect protein isoforms in a highly reproducible manner. The implications of this technique extend towards the diagnosis of many diseases because it can measure affected proteins or their isoforms.Though this method can provide insight into cell-signaling pathways, it can be also be applied to other systems where specific proteins need to be analyzed. Visual demonstration of this method is critical as the assay steps are difficult to learn because of the tricks, such as removing bubbles, loading the plate, et cetera. First, prepare a sample diluent mix by adding one microliter of DMSO inhibitor mix and two microliters of protease inhibitors to 47 microliters of sample diluent, so that the final concentration of DMSO and protease inhibitors is one X.Dilute previously isolated protein lysates using the sample diluent mix to obtain the desired concentrations.Next, add 3.325 microliters of standard ladder, six microliters of protease inhibitor, and three microliters of DMSO inhibitor to 137.675 microliters of antholite premix. Vortex the sample at least 15 seconds and keep on ice. Mix the two prepared solutions in a one to three ratio so that the final concentrations of DMSO protease inhibitors and the isoelectric point standard ladder are one X and the proteins in the capillary reach the final desired concentrations.After the proteins are added to the sample mixture, all the steps will be performed on ice or at four degrees Celsius. Place a 384-well plate on ice. Load ten microliters of the sample mix into the appropriate well of the plate, according to the assay template layout designed with the automated Western blotting system software.Following this, dilute the primary and secondary antibodies with antibody diluent. Then, pipette ten microliters of primary antibodies and 15 microliters of secondary antibodies into each well. Next, mix luminol and peroxide XDR in a one to one ratio.Pipette 15 microliters of the luminol peroxide mix into each well. Once the samples and reagants have been added to the plate, centrifuge for ten minutes at 2500 times G and four degrees Celsius to spin down the liquid and remove the bubbles. If bubbles still exist, remove them manually using a thin pipette tip.Open the automated Western blotting system software and click new'from the file menu. Go to the layout pane and assign locations for reagants and samples in the 384-well plate. Make a reagant assignment by clicking a well anywhere in the block.Select a row block of 12 wells each. Next, go to the layout pane tool bar and insert a sample, primary, secondary or luminol row block. Go to the protocol pane and select a reagant location in the plate.Then, click the cell in the sample column and select the reagant from the drop-down menu. In the same fashion, select the reagant locations for the primary, secondary, and luminol for each cycle. Click the cells one at a time in the primary or secondary antibody column and change the incubation times if necessary.Then, add cycles by clicking the Add'button in the protocol section. In the template pane, enter information pertaining to the sample, such as the identity of the primary and secondary antibodies, their catalog numbers and dilutions. Save the new assay file.Before clicking the start'button, quickly check the layout to make sure that everything is correct. Now, click start'to begin the run. The software will prompt the user to remove the waste, to refill the water and capillary box, to add anolite and catholite to the resource tray, to add wash buffer, and to load the plate into the cooled sample tray.An instrument status bar will be displayed on the computer screen a couple of minutes after the run has started. Click on the run summary'screen to see the status and separation panes. To observe the electrophoresis separation in a capillary, play the separation movie for that capillary by clicking the desired cycle and then clicking the play button in the control panel.Open the run file and select the analysis screen tab. Click edit'and then analysis'to change the isoelectric point range. Apply the appropriate isoelectric point standard.Add a peak fit, and add a peak name. Finally, select the data to use in the peaks tab and export the data for further analysis. The electropherogram of phosphorylated extracellular signal-regulated kinases from vascular endothelial growth-factor stimulated lysates is shown here.There's very high induction of phosphorylated proteins observed with vascular endothelial growth factor. The inset shows the endogenous loading control, HSP 70, indicating similar loading of samples for both untreated and treated samples. In a conventional immunoblot, only two bands were detected, even though the phosphorylated extracellular signal-regulated kinase proteins were resolved into four peaks by capillary isoelectric focusing analysis.The electropherogram of extracellular signal-regulated kinases from vascular endothelial growth-factor stimulated lysates is shown here. The inset shows the same loading control used in parallel. A conventional immunoblot shows only two bands corresponding to extracellular signal-regulated kinase one and extracellular signal-regulated kinase two.Whereas with the capillary isoelectric focusing, the extracellular signal-regulated kinase proteins were resolved into six peaks, corresponding to all four different phosphorylated peaks and two un-phosphorylated peaks. Once mastered, this technique can be done in a few hours, depending on the number of cycles, if it is performed properly. While attempting this procedure, it is important to remember to use higher antibody concentrations as compared to conventional immunoblots.Visual demonstration of this method is critical as the assay steps are difficult to learn because of tricks, such as removing bubbles, loading the plate, et cetera. After watching this video, you should have a good understanding of how to design an assay, prepare samples, run the assay, and analyze the data to observe different isoforms of your protein.

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