DESCRIPTION

HCN4 (Hyperpolarization-activated Cyclic Nucleotide-gated Channel 4) belongs to the potassium channel HCN family. It is a hyperpolarization-activated ion channel with very slow activation and inactivation, exhibiting weak selectivity for potassium over sodium ions. HCN4 may contribute to the native pacemaker currents in the heart (If) and in neurons (Ih). It is regulated by cyclic nucleotides such as cAMP, which modulate channel activity by shifting the voltage dependence of activation and altering the rate of channel opening and closing. HCN4 may mediate responses to sour stimuli. Defects in HCN4 are a cause of sick sinus syndrome type 2 (SSS2).

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with HRP) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with HRP also binds to the analyte. TMB acts as the HRP substrate and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns solution yellow. The color intensity is proportional to the quantity of bound protein which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 8 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 16 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	8	482.6	13.7	2.8
2	8	122.1	3.0	2.5
3	8	64.7	1.1	1.7

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	498.6	25.2	5.1
2	16	123.7	4.5	3.6
3	16	63.2	3.0	4.7

RECOVERY

The recovery of human/mouse/rat HCN4 spiked to three different levels throughout the range of the assay in cell lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Lysate	1:8	88	81-96
	1:16	104	93-115

LINEARITY

To assess the linearity of the assay, samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The tissue lysate was initially diluted 1:16. )

		Cell lysate	Tissue lysate
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	89	86
	Range (%)	88-90	84-89
1:8	Average% of Expected	90	97
	Range (%)	82-97	97-98
1:16	Average% of Expected	78	121
	Range (%)	77-79	119-123

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human/mouse/rat HCN4 produced at Sunlong Medical™.

Ordering & shipping

• Minimum order: 1 kit
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