Human AMD1 / S-adenosylmethionine decarboxylase proenzyme ELISA Kit
- SKU:
- HUFI00697
Description
Human AMD1 / S-adenosylmethionine decarboxylase proenzyme ELISA Kit - Information
The ELISA Genie AMD1 / S-adenosylmethionine decarboxylase proenzyme ELISA Kit can assay for AMD1 / S-adenosylmethionine decarboxylase proenzyme in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.
How our AMD1 / S-adenosylmethionine decarboxylase proenzyme 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 AMD1 / S-adenosylmethionine decarboxylase proenzyme 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 AMD1 / S-adenosylmethionine decarboxylase proenzyme ELISA Kit - Data
| Description | Essential for biosynthesis of the polyamines spermidine and spermine. Promotes maintenance and self-renewal of embryonic stem cells, by maintaining spermine levels. | |
| Post-Translational Modification | Is synthesized initially as an inactive proenzyme. Formation of the active enzyme involves a self-maturation process in which the active site pyruvoyl group is generated from an internal serine residue via an autocatalytic post-translational modification. Two non-identical subunits are generated from the proenzyme in this reaction, and the pyruvate is formed at the N-terminus of the alpha chain, which is derived from the carboxyl end of the proenzyme. The post-translation cleavage follows an unusual pathway, termed non-hydrolytic serinolysis, in which the side chain hydroxyl group of the serine supplies its oxygen atom to form the C-terminus of the beta chain, while the remainder of the serine residue undergoes an oxidative deamination to produce ammonia and the pyruvoyl group blocking the N-terminus of the alpha chain. | |
| Uniprot ID | P17707 |
| Alias | AMD1/AMD(S-adenosylmethionine decarboxylase proenzyme)/AdoMetDC/SAMDC | ||||||||||||||||||||
| Detection method | Sandwich ELISA Double Antibody | ||||||||||||||||||||
| Application | This immunoassay kit allows for the in vitro quantitative determination of AMD1 concentrations in serum plasma and other biological fluids. | ||||||||||||||||||||
| Size | 96T | ||||||||||||||||||||
| Range | 0.625-40ng/ml | ||||||||||||||||||||
| Sensitivity | < 0.375ng/ml | ||||||||||||||||||||
| Storage | 4'C for 6 months | ||||||||||||||||||||
| Recovery | Matrices listed below were spiked with certain level of AMD1 and the recovery rates were calculated by comparing the measured value to the expected amount of AMD1 in samples.
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| Linearity | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of AMD1 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% | ||||||||||||||||||||
| Note | For Research Use Only |
Human AMD1 / S-adenosylmethionine decarboxylase proenzyme ELISA Kit Protocol
The below protocol is a sample protocol for Human AMD1 / S-adenosylmethionine decarboxylase proenzyme 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 AMD1 / S-adenosylmethionine decarboxylase proenzyme 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
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 AMD1 / S-adenosylmethionine decarboxylase proenzyme ELISA Kit components | 96 Assays | 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:
The ELISA Genie Human AMD1 / S-adenosylmethionine decarboxylase proenzyme 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 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. |
Human AMD1 / S-adenosylmethionine decarboxylase proenzyme ELISA Kit Protein Information
| UniProt Protein Function: | AMD1: an important intermediate enzyme in polyamine biosynthesis. The polyamines spermine, spermidine, and putrescine are low-molecular-weight aliphatic amines essential for cellular proliferation and tumor promotion. Two alternatively spliced transcript variants that encode different proteins have been identified. Pseudogenes of this gene are found on chromosomes 5, 6, 10, X and Y.[provided by RefSeq, Jul 2010] |
| UniProt Protein Details: | Protein type:Amino Acid Metabolism - arginine and proline; Lyase; Amino Acid Metabolism - cysteine and methionine; EC 4.1.1.50 Chromosomal Location of Human Ortholog: 6q21 Cellular Component: cytosol Molecular Function:adenosylmethionine decarboxylase activity Biological Process: S-adenosylmethioninamine biosynthetic process; spermidine biosynthetic process; spermine biosynthetic process; polyamine metabolic process |
| NCBI Summary: | This gene encodes an important intermediate enzyme in polyamine biosynthesis. The polyamines spermine, spermidine, and putrescine are low-molecular-weight aliphatic amines essential for cellular proliferation and tumor promotion. Multiple alternatively spliced transcript variants have been identified. Pseudogenes of this gene are found on chromosomes 5, 6, 10, X and Y. [provided by RefSeq, Dec 2013] |
| UniProt Code: | P17707 |
| NCBI GenInfo Identifier: | 116241324 |
| NCBI Gene ID: | 262 |
| NCBI Accession: | P17707.2 |
| UniProt Secondary Accession: | P17707,Q5VXN4, Q5VXN6, Q9BWK4, E1P5F7, |
| UniProt Related Accession: | P17707 |
| Molecular Weight: | 21,301 Da |
| NCBI Full Name: | S-adenosylmethionine decarboxylase proenzyme |
| NCBI Synonym Full Names: | adenosylmethionine decarboxylase 1 |
| NCBI Official Symbol: | AMD1 |
| NCBI Official Synonym Symbols: | AMD; SAMDC; ADOMETDC |
| NCBI Protein Information: | S-adenosylmethionine decarboxylase proenzyme; S-adenosylmethionine decarboxylase 1 |
| UniProt Protein Name: | S-adenosylmethionine decarboxylase proenzyme |
| Protein Family: | S-adenosylmethionine decarboxylase proenzyme |
| UniProt Gene Name: | AMD1 |
| UniProt Entry Name: | DCAM_HUMAN |
