Human PRKG1 ELISA Kit
- SKU:
- HUFI02785
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- Q13976
- Sensitivity:
- 0.094ng/ml
- Range:
- 0.156-10ng/ml
- ELISA Type:
- Sandwich
- Synonyms:
- PRKG1, cGMP-dependent protein kinase I, cGKI, cGK 1
- Reactivity:
- Human
- Research Area:
- Cell Biology
Description
Human PRKG1 ELISA
PRKG1 (Protein Kinase CGMP-Dependent 1) is present in almost all types of smooth muscle, platelets, cerebellar Purkinje cells, hippocampal neurons, and the lateral amygdala. The PRKG1 proteins are also involved in cardiovascular and neurological function, as well as smooth muscle tone, platelet aggregation, and cell growth regulation. Aortic aneurysm, familial thoracic 8 and familial thoracic aortic aneurysm, and aortic dissection are diseases that are all linked to PRKG1. Sweet taste signalling and the activation of angiotensin II formation via ERK are among the pathways influenced by PRKG1. PRKG1 gene ontology (GO) annotations include transferase activity, the transferring of phosphorus-containing groups, and protein tyrosine kinase activity. PRKG2 is a paralog of PRKG1.
system_update_altDatasheet - तकनीकी मैनुअलsystem_update_altMSDS - तकनीकी मैनुअल
| Product Name: | Human PRKG1 ELISA Kit |
| Product Code: | HUFI02785 |
| Size: | 96 Assays |
| Alias: | PRKG1, cGMP-dependent protein kinase I, cGKI, cGK 1 |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human PRKG1 concentrations in serum plasma and other biological fluids. |
| Sensitivity: | 0.094ng/ml |
| Range: | 0.156-10ng/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Human PRKG1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human PRKG1 in samples. | ||||||||||||||||
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PRKG1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. | ||||||||||||||||
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| CV(%): | Intra-Assay: CV<8% Inter-Assay: CV<10% |
| Component | Quantity | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | 4°C for 6 months |
| Lyophilized Standard | 2 | 4°C/-20°C |
| Sample/Standard Dilution Buffer | 20ml | 4°C |
| Biotin-labeled Antibody(Concentrated) | 120ul | 4°C (Protect from light) |
| Antibody Dilution Buffer | 10ml | 4°C |
| HRP-Streptavidin Conjugate(SABC) | 120ul | 4°C (Protect from light) |
| SABC Dilution Buffer | 10ml | 4°C |
| TMB Substrate | 10ml | 4°C (Protect from light) |
| Stop Solution | 10ml | 4°C |
| Wash Buffer(25X) | 30ml | 4°C |
| Plate Sealer | 5 | - |
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
| Uniprot | Q13976 |
| UniProt Protein Function: | PKG1: an AGC kinase of the PKG family. Type I cGMP-dependent protein kinase; relaxes vascular smooth muscle and inhibits platelet aggregation. Binding of cGMP results in enzyme activation. Two splice variant isoforms have been described. |
| UniProt Protein Details: | Protein type:AGC group; EC 2.7.11.12; Kinase, protein; PKG family; Protein kinase, AGC; Protein kinase, Ser/Thr (non-receptor) Chromosomal Location of Human Ortholog: 10q11.23-q21.1 Cellular Component: cytoplasm; cytosol; plasma membrane Molecular Function:calcium channel regulator activity; cGMP-dependent protein kinase activity; protein binding; protein serine/threonine kinase activity Biological Process: actin cytoskeleton organization and biogenesis; regulation of GTPase activity; signal transduction |
| NCBI Summary: | Mammals have three different isoforms of cyclic GMP-dependent protein kinase (Ialpha, Ibeta, and II). These PRKG isoforms act as key mediators of the nitric oxide/cGMP signaling pathway and are important components of many signal transduction processes in diverse cell types. This PRKG1 gene on human chromosome 10 encodes the soluble Ialpha and Ibeta isoforms of PRKG by alternative transcript splicing. A separate gene on human chromosome 4, PRKG2, encodes the membrane-bound PRKG isoform II. The PRKG1 proteins play a central role in regulating cardiovascular and neuronal functions in addition to relaxing smooth muscle tone, preventing platelet aggregation, and modulating cell growth. This gene is most strongly expressed in all types of smooth muscle, platelets, cerebellar Purkinje cells, hippocampal neurons, and the lateral amygdala. Isoforms Ialpha and Ibeta have identical cGMP-binding and catalytic domains but differ in their leucine/isoleucine zipper and autoinhibitory sequences and therefore differ in their dimerization substrates and kinase enzyme activity. [provided by RefSeq, Sep 2011] |
| UniProt Code: | Q13976 |
| NCBI GenInfo Identifier: | 6225588 |
| NCBI Gene ID: | 5592 |
| NCBI Accession: | Q13976.3 |
| UniProt Secondary Accession: | Q13976,P14619, Q5JP05, Q5JSJ6, Q6P5T7, A5YM56, B3KSF3 E2PU10, |
| UniProt Related Accession: | Q13976 |
| Molecular Weight: | |
| NCBI Full Name: | cGMP-dependent protein kinase 1 |
| NCBI Synonym Full Names: | protein kinase, cGMP-dependent, type I |
| NCBI Official Symbol: | PRKG1 |
| NCBI Official Synonym Symbols: | PKG; cGK; AAT8; cGK1; cGKI; cGK 1; PRKG1B; PRKGR1B; cGKI-BETA; cGKI-alpha |
| NCBI Protein Information: | cGMP-dependent protein kinase 1 |
| UniProt Protein Name: | cGMP-dependent protein kinase 1 |
| UniProt Synonym Protein Names: | cGMP-dependent protein kinase I; cGKI |
| UniProt Gene Name: | PRKG1 |
*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.
| Step | 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. |
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 Type | Protocol |
| 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. |
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