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

Summary

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.

Abstract

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.

Introduction

Capillary isoelectric focusing (cIEF) is an automated, capillary-based immunoassay that resolves proteins on the basis of their charge^1,2,3. 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 b....

Protocol

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.

1. 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 µg/mL), and ascorbic acid (1 µg/mL).
2. Starve cells overnight....

Results

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.......

Discussion

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 reports^5,6,7

Materials

List of materials used in this article

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