Human PKD2 / Polycystin 2 ELISA Kit

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HUFI01404
€599

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Human PKD2 / Polycystin 2 ELISA Kit - Information

The ELISA Genie PKD2 / Polycystin 2 ELISA Kit can assay for PKD2 / Polycystin 2 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our PKD2 / Polycystin 2 ELISA Kits Work?

The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound PKD2 / Polycystin 2 is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Human PKD2 / Polycystin 2 ELISA Kit - Data

Description

Functions as a cation channel involved in fluid-flow mechanosensation by the primary cilium in renal epithelium (PubMed:18695040). Functions as outward-rectifying K+ channel, but is also permeable to Ca2+, and to a much lesser degree also to Na+ (PubMed:11854751, PubMed:15692563, PubMed:27071085, PubMed:27991905). May contribute to the release of Ca2+ stores from the endoplasmic reticulum (PubMed:11854751, PubMed:20881056). Together with TRPV4, forms mechano- and thermosensitive channels in cilium (PubMed:18695040). PKD1 and PKD2 may function through a common signaling pathway that is necessary to maintain the normal, differentiated state of renal tubule cells. Acts as a regulator of cilium length, together with PKD1. The dynamic control of cilium length is essential in the regulation of mechanotransductive signaling. The cilium length response creates a negative feedback loop whereby fluid shear-mediated deflection of the primary cilium, which decreases intracellular cAMP, leads to cilium shortening and thus decreases flow-induced signaling. Also involved in left-right axis specification via its role in sensing nodal flow; forms a complex with PKD1L1 in cilia to facilitate flow detection in left-right patterning. Detection of asymmetric nodal flow gives rise to a Ca2+ signal that is required for normal, asymmetric expression of genes involved in the specification of body left-right laterality.

Post-Translational Modification

Phosphorylated. Phosphorylation is important for protein function; a mutant that lacks the N-terminal phosphorylation sites cannot complement a zebrafish pkd2-deficient mutant (PubMed:16551655). PKD-mediated phosphorylation at the C-terminus regulates its function in the release of Ca2+ stores from the endoplasmic reticulum (PubMed:20881056). PKA-mediated phosphorylation at a C-terminal site strongly increases the open probability of the channel, but does not increase single channel conductance (PubMed:26269590). N-glycosylated. The four subunits in a tetramer probably differ in the extent of glycosylation; simultaneous glycosylation of all experimentally validated sites would probably create steric hindrance. Thus, glycosylation at Asn-305 is not compatible with glycosylation at Asn-328; only one of these two residues is glycosylated at a given time.

Uniprot ID Q13563
Alias

PKD2/Polycystin-2/Polycystwin/Autosomal dominant polycystic kidney disease type II protein/Polycystic kidney disease 2 protein

Detection method

Sandwich ELISA Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of PKD2 concentrations in serum plasma and other biological fluids.

Size

96T

Range

0.312-20ng/ml

Sensitivity

< 0.188ng/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of PKD2 and the recovery rates were calculated by comparing the measured value to the expected amount of PKD2 in samples.

Matrix Recovery range(%) Average(%)
serum(n=5) 86-98 91
EDTA plasma(n=5) 85-105 95
UFH plasma(n=5) 88-102 94
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of PKD2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 91-101% 87-102% 92-99% 87-103%
EDTA plasma(n=5) 83-101% 84-100% 83-101% 84-99%
UFH plasma(n=5) 90-98% 80-97% 85-100% 85-97%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Human PKD2 / Polycystin 2 ELISA Kit Protocol

The below protocol is a sample protocol for Human PKD2 / Polycystin 2 ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human PKD2 / Polycystin 2 present in their sample.

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.

Sandwich Protocol

Sandwich ELISA Protocol

Kit Protocol:

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.

Human PKD2 / Polycystin 2 ELISA Kit components

96 Assays

Storage

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:

The ELISA Genie Human PKD2 / Polycystin 2 ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • 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

Sample Preparation

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

Serum

If 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.

Plasma

Collect 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 Fluid

Collect 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 supernatant

Collect 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 lysates

Solubilize 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 homogenates

The 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 lysates

Rinse 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 Milk

Collect 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.

Human PKD2 / Polycystin 2 ELISA Kit Protein Information

UniProt Protein Function:PKD2: Involved in fluid-flow mechanosensation by the primary cilium in renal epithelium. PKD1 and PKD2 may function through a common signaling pathway that is necessary for normal tubulogenesis. Acts as a regulator of cilium length, together with PKD1. The dynamic control of cilium length is essential in the regulation of mechanotransductive signaling. The cilium length response creates a negative feedback loop whereby fluid shear-mediated deflection of the primary cilium, which decreases intracellular cAMP, leads to cilium shortening and thus decreases flow-induced signaling. Functions as a calcium permeable cation channel. Defects in PKD2 are the cause of polycystic kidney disease autosomal dominant type 2 (ADPKD2). ADPKD2 is a disorder characterized by progressive formation and enlargement of cysts in both kidneys, typically leading to end-stage renal disease in adult life. Cysts also occurs in the liver and other organs. It represents approximately 15% of the cases of autosomal dominant polycystic kidney disease. ADPKD2 is clinically milder than ADPKD1 but it has a deleterious impact on overall life expectancy. Belongs to the polycystin family. 5 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Membrane protein, integral; Channel, cation; Membrane protein, multi-pass

Chromosomal Location of Human Ortholog: 4q22.1

Cellular Component: filamentous actin; endoplasmic reticulum membrane; nonmotile primary cilium; endoplasmic reticulum; integral to plasma membrane; lamellipodium; cytoplasm; basal plasma membrane; plasma membrane; motile primary cilium; intercellular junction; basal cortex; cilium

Molecular Function:voltage-gated cation channel activity; actinin binding; identical protein binding; protein homodimerization activity; phosphoprotein binding; voltage-gated ion channel activity; calcium ion binding; muscle alpha-actinin binding; ATPase binding; calcium-induced calcium release activity; voltage-gated calcium channel activity; protein binding; potassium channel activity; voltage-gated sodium channel activity; cytoskeletal protein binding; HLH domain binding; receptor binding

Biological Process: neural tube development; positive regulation of inositol-1,4,5-triphosphate receptor activity; embryonic placenta development; cytoplasmic sequestering of transcription factor; positive regulation of nitric oxide biosynthetic process; regulation of cAMP metabolic process; heart development; detection of mechanical stimulus; JAK-STAT cascade; liver development; G1/S-specific positive regulation of cyclin-dependent protein kinase activity; regulation of cell proliferation; negative regulation of cell proliferation; ureteric bud branching; spinal cord development; release of sequestered calcium ion into cytosol; calcium ion transport; positive regulation of transcription from RNA polymerase II promoter; heart looping; cell cycle arrest; determination of left/right symmetry; centrosome duplication

Disease: Polycystic Kidney Disease 2

NCBI Summary:This gene encodes a member of the polycystin protein family. The encoded protein is a multi-pass membrane protein that functions as a calcium permeable cation channel, and is involved in calcium transport and calcium signaling in renal epithelial cells. This protein interacts with polycystin 1, and they may be partners in a common signaling cascade involved in tubular morphogenesis. Mutations in this gene are associated with autosomal dominant polycystic kidney disease type 2. [provided by RefSeq, Mar 2011]
UniProt Code:Q13563
NCBI GenInfo Identifier:116242717
NCBI Gene ID:5311
NCBI Accession:Q13563.3
UniProt Secondary Accession:Q13563,O60441, Q15764, Q2M1Q3, Q2M1Q5,
UniProt Related Accession:Q13563
Molecular Weight:968
NCBI Full Name:Polycystin-2
NCBI Synonym Full Names:polycystic kidney disease 2 (autosomal dominant)
NCBI Official Symbol:PKD2  
NCBI Official Synonym Symbols:PC2; PKD4; Pc-2; APKD2; TRPP2  
NCBI Protein Information:polycystin-2; R48321; polycystwin; autosomal dominant polycystic kidney disease type II protein; transient receptor potential cation channel, subfamily P, member 2
UniProt Protein Name:Polycystin-2
UniProt Synonym Protein Names:Autosomal dominant polycystic kidney disease type II protein; Polycystic kidney disease 2 protein; Polycystwin; R48321
Protein Family:Polycystin
UniProt Gene Name:PKD2  
UniProt Entry Name:PKD2_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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