DESCRIPTION

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are a group of voltage-gated ion channels in which the threshold voltage is modulated by the metabolic state of the cell (levels of cyclic nucleotides, like cAMP), thus allowing them to regulate cellular voltage in a context-specific manner. They open at hyperpolarized (negative) Vmem, giving rise to currents that are a mix of sodium and potassium fluxes. Among the four HCN isoforms (HCN1-4), HCN2 channels show an especially high sensitivity to cAMP.

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	509.4	17.8	3.5
2	8	119.7	3.8	3.2
3	8	55.3	2.4	4.4

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	505.3	15.5	3.1
2	16	118.8	3.7	3.1
3	16	56.6	2.6	4.7

RECOVERY

The recovery of human/mouse/rat HCN2 spiked to three different levels throughout the range of the assay in tissue lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Tissue lysate	1:2,048	106	100-113

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 1:128.)

		Tissue lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	107
	Range (%)	104-110
1:8	Average% of Expected	115
	Range (%)	113-118
1:16	Average% of Expected	120
	Range (%)	119-120

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human/Mouse/Rat HCN2 Sandwich ELISA Kit produced at Sunlong Medical™.

Ordering & shipping

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