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 (10 ng/mL), insulin-like growth factor (20 ng/mL), hydrocortisone (0.2 &#181;g/mL), and ascorbic acid (1 &#181;g/mL).</li>
	<li>Starve cells overnight with endothelial cell basal medium supplemented with 1% FCS and no growth factor supplement.</li>
	<li>Aspirate all medium, add 2 mL of endothelial cell culture medium without growth factors (starvation medium) in one dish (control), then add 50 ng/mL VEGF in 2 mL of starvation culture medium in a second dish for 7 min.</li>
	<li>Aspirate the medium and wash cells 2 times with 10 mL room temperature PBS.</li>
	<li>Ensure the Petri dishes are on ice and keep them there from this step onwards.</li>
	<li>Add 250-400 &#181;L of ice cold lysis buffer (Bicine/CHAPS; see Table of Materials) containing aqueous protease inhibitor mix and DMSO inhibitor mix (Phosphatase inhibitors; see Table of Materials) to each 10 cm plate. Swirl the plate to ensure proper coverage and keep it for 10 min on ice. 
	NOTE: The final concentration of protease and the DMSO inhibitor mix should be 1x.</li>
	<li>Scrape the cells from the dish with a cell scraper and transfer to a pre-chilled microfuge tube. Pipette up and down 5 times to lyse cells.</li>
	<li>Briefly sonicate the cells at 4 &#176;C. Set the sonicator as follows: number of cycles = 5, power = LOW, ON = 5 sec, OFF = 30 s. This step is included to break nucleic acids, and not to lyse cells. 
	NOTE: Sonication should be mild to prevent denaturation of proteins.</li>
	<li>Vortex the tube for 5 s (not continuously), 2 s at a time (3 times), and keep on ice.</li>
	<li>Clarify lysate by centrifugation at 14,000 x g for 15 min at 4 &#176;C, then immediately transfer the supernatant to a clean pre-chilled microfuge tube.</li>
	<li>Measure the protein concentration using a Bicinchoninic Acid (BCA) protein assay kit4.</li>
	<li>Make 10 &#181;L aliquots, snap freeze in dry ice or liquid nitrogen, and store at -80 &#176;C until use.</li>
</ol>
2. Sample Mix Preparation (Calculation for 150 &#181;L Sample Mix)
<ol>
	<li>First, prepare a sample diluent mix by adding 1 &#181;L of DMSO inhibitor mix (stock 50x) and 2 &#181;L of protease inhibitors (stock 25x) to 47 &#181;L of sample diluent (see Table of Materials), so that the final concentration of DMSO inhibitors and protease inhibitors becomes 1x. Next, dilute the protein lysates using the sample diluent mix to obtain the desired concentrations (see step 2.3).</li>
	<li>Prepare ampholyte/ladder/protease inhibitor/DMSO inhibitor mix by adding 3.325 &#181;L standard ladder (see Table of Materials; stock 60x), 6 &#181;L of protease inhibitor (25x), 3 &#181;L of DMSO (50x) inhibitor to 137.675 &#181;L ampholyte premix (see Table of Materials). Vortex the tube at least 15 s total, 5 s at a time (3-4 times), and keep on ice.</li>
	<li>Mix the solutions from steps 2.1 and 2.2 in a 1:3 ratio, so that the final concentrations of DMSO, protease inhibitors, and the pI standard ladder become 1X, and the proteins in the capillary reach the final desired concentrations (e.g., 50 &#181;g/mL was used for Figure 2). 
	NOTE: Protein concentration in the capillary and well are the same.</li>
	<li>Load 10 &#181;L of sample mix into the appropriate well, according to the 384-well plate assay template layout (see step 3 and Figure 1) and keep the plate on ice.</li>
	<li>Dilute the primary (pERK1/2, 1:50; ERK1/2, 1:100; HSP 70, 1:500) and secondary antibodies (1:300) with antibody diluent (see Table of Materials) and pipette 10 &#181;L of primary and 15 &#181;L of secondary antibodies into each well. 
	NOTE: In general, higher concentration of antibodies are required for cIEF assays as compared to conventional western blots.</li>
	<li>Mix Luminol and peroxide XDR (1:1 ratio; see Table of Materials) together and pipette 15 &#181;L into each well. 
	NOTE: Mix these solutions fresh every time before use and keep on ice.</li>
	<li>Once samples and all reagents are pipetted into the plate, centrifuge the plate at 2,500 x g for 10 min at 4 &#176;C to spin down the liquid and remove the bubbles. If bubbles still exist, remove them manually using a thin pipette tip. 
	NOTE: Do not load more than 20 &#181;L of sample or reagents per well; otherwise, the instrument pipette cannot be washed properly. Make sure to follow the instruction manual from the manufacturer to prepare all reagents.</li>
</ol>
3. Designing a New Assay Template with System Software (Figure 1)
<ol>
	<li>Open the system software and click &#34;new&#34; from the File menu.</li>
	<li>Go to the layout pane and assign locations for reagents and samples in a 384 well-plate. Use up to a maximum of 96 wells per 384-well plate.</li>
	<li>Make a reagent assignment by clicking a well anywhere in the block.Select a row block of 12 wells each. Wells, e.g., from 1-12 or 13-24, are assigned as a block by default.</li>
	<li>Go to the layout pane tool bar and insert either an empty row block or a sample, primary, secondary, or luminol row block. Each block is assigned a different color, so they are easy to differentiate. 
	NOTE: When clicking a well, a black border can be seen around the block where the new block will be inserted in the pane. A row block can also be deleted.</li>
	<li>Go to the protocol pane and select a reagent location in the plate. Then, click a cell in the sample column and select the reagent from the dropdown menu.</li>
	<li>In this same fashion, select the reagent locations for the primary, secondary, and luminol for each cycle.</li>
	<li>Click the cells, one at a time, in the primary or secondary antibody column, and change the incubation times, if necessary. 
	NOTE: Entry notes for each cycle can also be added.</li>
	<li>Add cycles by clicking the &#39;add&#39; button in the protocol section. 1 cycle, 4 cycles, or all cycles&#160;(a maximum of 8 cycles can be added to the protocol). 
	NOTE: It is possible to copy and paste cycle information in this step: select a cycle, go to edit, and copy and paste.</li>
	<li>Enter information pertaining to the sample, such as the identity of the primary and secondary antibodies, their catalog numbers and dilutions, etc., in the template pane. This is an optional step that is useful during the post-run analysis.</li>
	<li>Save the new assay file. Before clicking the &#39;start&#39; button, quickly check the layout to make sure that everything is correct.</li>
</ol>
4. Protocol for cIEF Instrument Setting
<ol>
	<li>Set capillary isoelectric focusing electrophoresis separation conditions. These should be 15,000 &#181;W and 40 min, and the UV immobilization time should be 80 s. However, the UV immobilization time can be set within the range of 80-140 s, and may need to be optimized for the protein of interest. 
	NOTE: A time point for UV immobilization can be set for each cycle, but not for each capillary.</li>
	<li>Set wash 1 to 2 washes, for 150 s each wash (default), and primary antibody incubation for 120 min.</li>
	<li>Set wash 2 to 2 washes, for 150 s each wash (default), and secondary antibody incubation for 60 min.</li>
	<li>Set wash 3, which should be 2 washes, for 150 s each wash (default).</li>
	<li>Set exposure times when chemiluminescence will be detected; these should be 30, 60, 120, 240, 490, and 960 s. All steps (1-8) will be carried out in a single capillary.</li>
</ol>
5. Running the Assay File
<ol>
	<li>Once everything is ready, click start to begin the run. The software will prompt you to remove the waste, to refill the water and the capillary box, to add anolyte and catholyte to the resource tray, to add wash buffer and finally to load the plate into the cooled sample tray. 
	NOTE: Remove the lid from the capillary box, but do not remove the lid from the assay plate. The lid from the plate can be removed automatically by the instrument when necessary. This helps to prevent reagent evaporation.</li>
	<li>A couple of minutes after the run has been started, an instrument status bar will be displayed on the computer screen. Status messages and progress bars can be seen corresponding to the instrument&#39;s current stage. 
	NOTE: Initially, the instrument will check if everything is correct before proceeding to pipette the anolyte and catholyte into the separation tray, picking the first block of 12 capillaries, removing the lid of the plate, and placing the samples from the sample plate into the capillaries, and then finally into the separation chamber for electrophoresis.</li>
	<li>Click on the run summary screen to see two panes: status and separation. Users can view the run progress, and movies of the separation (the 12 capillaries of each cycle) can be stored when the electrophoresis step of each cycle is completed. To observe the electrophoresis separation in a capillary, play the separation movie for that capillary by clicking the desired cycle and then the play button in the control panel.</li>
	<li>The run file can be stored automatically upon the completion of the run.</li>
</ol>
6. Analyze the Data (Figure S1)
<ol>
	<li>Open the run file and select the analysis screen tab. In Figure S1A, the data shown (peaks) in the sample graph is from the selected capillary (i.e., 4th capillary from cycle 1).</li>
	<li>Click edit and then analysis (Figure S1B). At this stage, users can change the pI range, apply the appropriate pI standard for a given experiment, add a peak fit, and add a peak name. 
	NOTE: When using a pH 5-8 ampholyte premix (as is the case here), the recommended standard ladder to use is one with fluorescently labeled peptides with pIs of 4.9, 6.0, 6.4, 7.0, and 7.3, and when using a pH 3-10 premix, the recommended ladder is one with pIs of 4.0, 4.9, 6.0, 6.4, and 7.3.2</li>
	<li>Results are displayed in the Peaks tab; the values displayed for the samples are Position, pI, peak Height and Area, % Area, peak Width, and signal to noise (S/N) (Figure S1C). Choose which data to use, and export the data for further analysis. 
	NOTE: Data can only be exported after labeling the peak (Figure S1C).</li>
</ol>

<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>

The author has no disclosures.

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

highly-sensitive-quantitative-detection-proteins-their-isoforms

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

6.6K Views.  751 85, Uppsala, Sweden. 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. 

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

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 biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules. Besides, the electro-driven separation in CZE is governed by extensive properties of matter and offers the advantage of analyzing proteins close to their native state. However, the successful implementation of this technique for routine analysis depends on the skills of the analyst at the critical steps during sample and system preparation. The purpose of this tutorial is to detail the steps to succeed in the CZE analysis of mAbs. Further, this protocol can be used for the development and improvement of skills of the personnel involved in protein analytical chemistry laboratories.

Here, we present a comprehensive capillary zone electrophoresis protocol for the assessment of intrinsic physicochemical heterogeneity of monoclonal antibodies as a quality attribute.

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

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

Chemistry

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules feature the presence of at least one energetic pyrophosphate moiety on the myo-inositol ring. They exist ubiquitously in eukaryotes and operate as metabolic messengers surveying phosphate homeostasis, insulin sensitivity, and cellular energy charge. Owing to the absence of a chromophore in these metabolites, a very high charge density, and low abundance, their analysis requires radioactive tracer, and thus it is convoluted and expensive. Here, the study presents a detailed protocol to perform absolute and high throughput quantitation of inositol pyrophosphates from mammalian cells by capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS). This method enables the sensitive profiling of all biologically relevant PP-InsPs species in mammalian cells, enabling baseline separation of regioisomers. Absolute cellular concentrations of PP-InsPs, including minor isomers, and monitoring of their temporal changes in HCT116 cells under several experimental conditions are presented.

A procedure for capillary electrophoresis electrospray ionization mass spectrometry for the absolute quantitation of inositol pyrophosphates from mammalian cell extracts is described.

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Biology

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method

Natural products derived from plants and microbes are a rich source of bioactive molecules. Prior to their use, the active molecules from complex extracts must be purified for downstream applications. There are various chromatographic methods available for this purpose yet not all labs can afford high performance methods and isolation from complex biological samples can be difficult. Here we demonstrate that preparative liquid-phase isoelectric focusing (IEF) can separate molecules, including small molecules and peptides from complex plant extracts, based on their isoelectric points (pI). We have used the method for complex biological sample fractionation and characterization. As a proof of concept, we fractionated a Gymnema sylvestre plant extract, isolating a family of terpenoid saponin small molecules and a peptide. We also demonstrated effective microbial protein separation using the Candida albicans fungus as a model system.

Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing IEF Method

The objective is to fractionate and isolate bioactive small molecules, peptides from a complex plant extract, and proteins from pathogenic microbes by employing liquid-phase isoelectric focusing (IEF) method. Further, the separated molecules were identified and their bioactivity confirmed.

Natural products derived from plants and microbes are a rich source of bioactive molecules. Prior to their use, the active molecules from complex extracts ...

Immunology and Infection

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and &#945;-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an &#945;-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...

Capillary Electrophoresis (CE)

Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Capillary electrophoresis (CE) is a separation technique that separates molecules in an electric field according to size and charge. CE is performed in a small glass tube called a capillary that is filled with an electrolyte solution. Analytes are separated due to differences in electrophoretic mobility, which varies with charge, solvent viscosity, and size. Traditional electrophoresis in gels is limited in the amount of voltage that can be applied because Joule heating effects will ruin the gel and the separation. Capillaries have a large surface area-to-volume ratio and thus dissipate heat better. Therefore, the voltages applied for a capillary electrophoresis experiment are quite large, often 10,000&#8211;20,000 V.
Capillary electrophoresis is useful for high-performance separations. Compared to liquid chromatography, CE separations are often faster and more efficient. However, capillary electrophoresis works best to separate charged molecules, which is not a limitation of liquid chromatography. CE has a greater peak capacity than high-performance liquid chromatography (HPLC), meaning the separations are more efficient and more peaks can be detected. The instrumentation can be very simple. However, HPLC is more versatile and many stationary and mobile phases have been developed for different types of molecules.

Capillary Electrophoresis: Principle, Procedure and Applications

Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Capillary electrophoresis (CE) is a separation technique that separates molecules in an ...

Education

JoVE Science Education

Analytical Chemistry

A Capillary Electrophoresis Immunoassay for Protein Biomarker Detection

Source: Sage, J. M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/60638">Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates a capillary electrophoresis immunoassay protocol for detecting a protein biomarker associated with amyotrophic lateral sclerosis (ALS) from human blood platelets. Confirmation of the presence of the ALS biomarker protein is achieved through size-based separation of the platelet proteins followed by chemiluminescent detection.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of buffers and ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

Electrophoresis: Overview

Electrophoresis is a powerful analytical separation technique that relies on the differential migration of charged species when subjected to an electric field. The core strength of electrophoresis lies in its ability to separate high-molecular-weight species in complex mixtures. It has found widespread use in biochemistry, molecular biology, and analytical chemistry, allowing the separation of compounds like amino acids, nucleotides, carbohydrates, and proteins with excellent resolution.
There are two main formats for electrophoretic separations: slab electrophoresis and capillary electrophoresis. Slab Electrophoresis uses a gel matrix, such as agarose or polyacrylamide, which acts as a sieve, allowing molecules to move through the pores. Capillary electrophoresis employs a narrow capillary tube filled with a buffer solution, providing high-resolution separation with rapid analysis time and minimal sample requirements. Although slab electrophoresis is widely used in biochemistry and biology to separate high-molecular-mass species, capillary electrophoresis is a more recent development with several advantages.
Capillary electrophoresis is a separation technique used to analyze complex mixtures of analytes based on their differential migration rates in an applied electric field. In this method, a sample is injected into a narrow capillary tube filled with a buffered solution, and an electric field is applied to facilitate separation. The migration rate of the analyte depends on its charge and size. The smaller, more highly charged ions migrate faster than the larger, less charged ones. In capillary electrophoresis, the migration of sample components is influenced by two types of mobility: electrophoretic mobility and electroosmotic mobility. Electrophoretic mobility is the solute&#39;s response to the applied electric field, with cations moving toward the negatively charged cathode, anions moving toward the positively charged anode, and neutral species remaining stationary. Electroosmotic mobility occurs when the buffer solution moves through the capillary in response to the electric field, typically toward the cathode.
Under normal conditions, capillary electrophoresis separates cations first, followed by neutral species, and finally, the anions. This separation is based on their respective electrophoretic mobilities and electroosmotic flow velocities. Notably, all separated species are eluted from the same end of the capillary, allowing their detection using quantitative detectors. The detector signals produce an electropherogram resembling a chromatogram but feature narrower peaks.
Capillary electrophoresis is a powerful tool for qualitative and quantitative analysis of complex mixtures. It provides information similar to that obtained from other separation techniques, such as gas chromatography (GC) or high-performance liquid chromatography (HPLC).

Electrophoresis is a powerful analytical separation technique that relies on the differential migration of charged species when subjected to an electric ...

JoVE Core

Unit 1

Principles of Chromatography

KEY TERMS AND CONCEPTS

Capillary Electrophoresis: Instrumentation

Capillary electrophoresis instrumentation typically consists of several key components. A high-voltage power supply generates the electric field necessary for the separation by connecting to an anode (the positively charged electrode) and a cathode (the negatively charged electrode) located in buffer reservoirs at each end of the capillary tube. The system includes a sample vial, a fused silica capillary tube coated with polyimide for mechanical strength through which the sample components migrate during separation, and a detector to analyze the separated components.
The sample must be introduced into the capillary tube to initiate the process. This is achieved by detaching one end of the capillary and its electrode from the associated buffer reservoirs and placing them in the sample vial. Sample introduction can be accomplished through hydrodynamic injection, which involves applying pressure to the sample vial, or electrokinetic injection, which relies on an electric field to drive the sample into the capillary.
When current flows through the capillary containing a conductive buffer solution, it leads to Joule heating due to the narrow bore of the capillary column and the relative thickness of the capillary&#39;s walls. Joule heating refers to the heat generated as an electric current passes through a conductive medium&#8212;in this case, the buffer solution inside the capillary. It is directly related to the energy dissipation in the form of heat, as defined by the equation Q = I&#178;Rt, where Q is the heat energy (in joules), I is the current, R is the resistance, and t is time. In Capillary electrophoresis, this heating can change the viscosity of the buffer solution, causing solutes in the center of the capillary to migrate faster than those near the walls, resulting in band broadening and degraded separation. Capillaries with smaller inner diameters generate less Joule heating, while those with larger outer diameters are more effective at dissipating heat.
A stacking technique may be employed to enhance detection sensitivity in cases where the sample concentration is low. This method involves injecting the sample into a solution with a lower ionic strength than the buffering solution, causing the sample components to concentrate at the interface between the two solutions.
Once the sample has been introduced, a high voltage is applied across the buffer system, prompting charged species to migrate toward the cathode through electroosmotic flow. The separation of components occurs based on their electrophoretic mobility within the electric field.
Various detectors, such as absorption, fluorescence, conductivity, and mass spectrometry, are commonly employed in capillary electrophoresis to detect and analyze the separated components.

Capillary electrophoresis instrumentation typically consists of several key components. A high-voltage power supply generates the electric field necessary ...