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 ...

Research

JoVE Journal

Biochemistry

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The ...

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Detection of Heterodimerization of Protein Isoforms Using an in Situ Proximity Ligation Assay

Regulated protein-protein interactions are a guiding principle for many signaling events, and the detection of such events is an important element in ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...