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Human HCN4 ELISA Kit

SKU:
HUFI01251
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q9Y3Q4
Sensitivity:
0.188ng/ml
Range:
0.313-20ng/ml
ELISA Type:
Sandwich
Synonyms:
HCN4, Potassium, sodium hyperpolarization-activated cyclic nucleotide-gated channel 4, hyperpolarization activated cyclic nucleotide-gated cation channel 4, hyperpolarization activated cyclic nucleotide-gated potassium channel 4
Reactivity:
Human
Research Area:
Signal Transduction
$719
Frequently bought together:

Description

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Human HCN4 ELISA Kit

The Human HCN4 (Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 4) ELISA Kit is a powerful tool for the accurate measurement of HCN4 levels in human samples such as serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit delivers precise and dependable results, making it a valuable asset for a variety of research applications.HCN4 is a critical protein involved in the regulation of heart function, specifically in the generation of pacemaker currents in the heart. It plays a vital role in the cardiac electrophysiology and is associated with conditions such as arrhythmias and heart failure.

By detecting and quantifying HCN4 levels, researchers can gain insights into the molecular mechanisms underlying these cardiovascular diseases and potentially identify new therapeutic targets.Overall, the Human HCN4 ELISA Kit offers researchers a reliable tool for studying the role of HCN4 in cardiovascular health and disease, providing valuable data for both basic research and clinical applications.

Product Name:Human HCN4 ELISA Kit
Product Code:HUFI01251
Size:96 Assays
Alias:HCN4, Potassium, sodium hyperpolarization-activated cyclic nucleotide-gated channel 4, hyperpolarization activated cyclic nucleotide-gated cation channel 4, hyperpolarization activated cyclic nucleotide-gated potassium channel 4
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human HCN4 concentrations in serum plasma and other biological fluids.
Sensitivity:0.188ng/ml
Range:0.313-20ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human HCN4 and the recovery rates were calculated by comparing the measured value to the expected amount of Human HCN4 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)87-10596
EDTA plasma(n=5)85-10396
UFH plasma(n=5)91-10398
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human HCN4 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)87-102%85-105%88-100%
EDTA plasma(n=5)83-101%85-90%85-101%
UFH plasma(n=5)83-99%84-94%82-98%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

Other materials and equipment required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir
UniprotQ9Y3Q4
UniProt Protein Function:HCN4: Hyperpolarization-activated ion channel with very slow activation and inactivation exhibiting weak selectivity for potassium over sodium ions. May contribute to the native pacemaker currents in heart (If) and in neurons (Ih). Activated by cAMP. May mediate responses to sour stimuli. Defects in HCN4 are a cause of sick sinus syndrome type 2 (SSS2); also known as atrial fibrillation with bradyarrhythmia or familial sinus bradycardia. The term 'sick sinus syndrome' encompasses a variety of conditions caused by sinus node dysfunction. The most common clinical manifestations are syncope, presyncope, dizziness, and fatigue. Electrocardiogram typically shows sinus bradycardia, sinus arrest, and/or sinoatrial block. Episodes of atrial tachycardias coexisting with sinus bradycardia ('tachycardia-bradycardia syndrome') are also common in this disorder. SSS occurs most often in the elderly associated with underlying heart disease or previous cardiac surgery, but can also occur in the fetus, infant, or child without heart disease or other contributing factors, in which case it is considered to be a congenital disorder. Defects in HCN4 are the cause of Brugada syndrome type 8 (BRGDA8). A tachyarrhythmia characterized by right bundle branch block and ST segment elevation on an electrocardiogram (ECG). It can cause the ventricles to beat so fast that the blood is prevented from circulating efficiently in the body. When this situation occurs (called ventricular fibrillation), the individual will faint and may die in a few minutes if the heart is not reset. Belongs to the potassium channel HCN family.
UniProt Protein Details:Protein type:Membrane protein, multi-pass; Membrane protein, integral; Channel, cation

Chromosomal Location of Human Ortholog: 15q24.1

Cellular Component: integral to membrane; intrinsic to plasma membrane; perinuclear region of cytoplasm; plasma membrane

Molecular Function:cAMP binding; identical protein binding; intracellular cAMP activated cation channel activity; voltage-gated potassium channel activity; voltage-gated sodium channel activity

Biological Process: blood circulation; cation transport; muscle contraction; regulation of heart rate; regulation of membrane potential; synaptic transmission

Disease: Brugada Syndrome 8; Sick Sinus Syndrome 2, Autosomal Dominant

NCBI Summary:This gene encodes a member of the hyperpolarization-activated cyclic nucleotide-gated potassium channels. The encoded protein shows slow kinetics of activation and inactivation, and is necessary for the cardiac pacemaking process. This channel may also mediate responses to sour stimuli. Mutations in this gene have been linked to sick sinus syndrome 2, also known as atrial fibrillation with bradyarrhythmia or familial sinus bradycardia. Two pseudogenes have been identified on chromosome 15. [provided by RefSeq, Oct 2008]
UniProt Code:Q9Y3Q4
NCBI GenInfo Identifier:38605641
NCBI Gene ID:10021
NCBI Accession:Q9Y3Q4.1
UniProt Secondary Accession:Q9Y3Q4,Q9UMQ7,
UniProt Related Accession:Q9Y3Q4
Molecular Weight:129,042 Da
NCBI Full Name:Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
NCBI Synonym Full Names:hyperpolarization activated cyclic nucleotide gated potassium channel 4
NCBI Official Symbol:HCN4  
NCBI Official Synonym Symbols:SSS2  
NCBI Protein Information:potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
UniProt Protein Name:Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
Protein Family:Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel
UniProt Gene Name:HCN4  
UniProt Entry Name:HCN4_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

StepProtocol
1.Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3.Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6.Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9.Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample TypeProtocol
SerumIf using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

PlasmaCollect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal FluidCollect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatantCollect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysatesSolubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenatesThe preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysatesRinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast MilkCollect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

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