A Capillary Electrophoresis Immunoassay for Protein Biomarker Detection

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. 

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