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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A Capillary Electrophoresis Immunoassay for Protein Biomarker Detection. J. Vis. Exp. (Pending Publication), e22134, doi: (2024).

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