A Capillary Electrophoresis Immunoassay for Protein Biomarker Detection
Published: April 30, 2024
Abstract
Source: Sage, J. M., et al. Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets. J. Vis. Exp. (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.
Protocol
All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines. 1. Preparation of buffers and reagents NOTE: Prepare all samples as per the manufacturer's guidelines. During this procedure, wear personal protection equipment (lab coats, gloves, and goggles). Prepare citrate wash buffer by combining 0.941 g of sucrose (11 mM final concentration), 6.4 mL of 5 M sodi…
Representative Results
Figure 1: Assay layout. Both primary antibody and target protein sample optimization can be performed in one assay. Capillaries 2–7, 8–13, 14–19, and 20–24 represent various protein concentrations and primary antibody ranges. Capillary 25 represents positive control. Anti-ERK antibody was used; however, any appropriate positive control can be included.
Divulgaciones
The authors have nothing to disclose.
Materials
| 12-230 kDa Separation kit | ProteinSimple | SM-W004 | Contains pre-filled assay plate and 25-channel capillary cartridge |
| 3000G Thermocycler | Techne | FTC3G/02 | We used this thermocylcer for heating the sample mix |
| Anti-Mouse detection kit | ProteinSimple | 042-205 | Includes HRP-conjugated secondary antibody, buffer, luminol reagent, molecular weight marker |
| Anti-P(S409-410) TDP-43 antibody | ProteinTech | 22309-1-AP | Primary antibody that recognizes phosphorylated TDP-43 |
| Anti-P(S409-412) TDP-43 antibody | CosmoBio-USA | TIP-PTD-P02 | Primary antibody that recognizes phosphorylated TDP-43 |
| Anti-Rabbit detection kit | ProteinSimple | DM-001 | Includes HRP-conjugated secondary antibody, buffer, luminol reagent, molecular weight marker |
| Anti-TDP-43 (pan) antibody | ProteinTech | 10782-2-AP | Primary antibody that recognizes whole TDP-43 protein |
| Sonic Dismembrator; Model100 | Fisher Scientific | Sonicator. Used to rupture the cell membrane. This model is discontinued (Model XL2000-350) | |
| Table top centrifuge | Eppendorf | 22625004 | Model# 5810 with swinging plate bucket |
| Wes analyzer | ProteinSimple | 55892-WS-2203 | Performs the capillary gel electrophoresis |
| Compass for SimpleWestern (SW) | ProteinSimple | Ver.4.0.0. | Compass for SW is the control and data analysis application for SimpleWestern instruments |
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Citar este artículo
A Capillary Electrophoresis Immunoassay for Protein Biomarker Detection. J. Vis. Exp. (Pending Publication), e22134, doi: (2024).