Human GC / Vitamin D Binding protein ELISA Kit
- SKU:
- HUFI02386
Description
Human GC / Vitamin D Binding protein ELISA Kit - Information
The ELISA Genie GC / Vitamin D Binding protein ELISA Kit can assay for GC / Vitamin D Binding protein in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.
How our GC / Vitamin D Binding protein 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 GC / Vitamin D Binding protein 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 GC / Vitamin D Binding protein ELISA Kit - Data
| Description | This transcriptional activator recognizes and binds to the sequence 5'-RTTAYGTAAY-3' found in the promoter of genes such as albumin, CYP2A4 and CYP2A5. It is not essential for circadian rhythm generation, but modulates important clock output genes. May be a direct target for regulation by the circadian pacemaker component clock. May affect circadian period and sleep regulation. | |
| Post-Translational Modification | ||
| Uniprot ID | Q10586 |
| Alias | DBP/Vitamin D Binding Protein/DBP-MAF/Gc/Gc-MAF/VDBG/VDBP/DBP/GC/DBPVDBG/EC 6.3.1.5/Gc-globulin/gc-globulin/GRD3/Group-specific component/group-specific component(vitamin D binding protein)/hDBP/VDB/VDBP/vitamin D-binding alpha-globulin/vitamin D-binding protein/ | ||||||||||||||||||||
| Detection method | Sandwich ELISA Double Antibody | ||||||||||||||||||||
| Application | This immunoassay kit allows for the in vitro quantitative determination of DBP/Vitamin D Binding Protein concentrations in serum plasma and other biological fluids. | ||||||||||||||||||||
| Size | 96T | ||||||||||||||||||||
| Range | 3.906-250ng/ml | ||||||||||||||||||||
| Sensitivity | < 2.344ng/ml | ||||||||||||||||||||
| Storage | 4'C for 6 months | ||||||||||||||||||||
| Recovery | Matrices listed below were spiked with certain level of DBP/Vitamin D Binding Protein and the recovery rates were calculated by comparing the measured value to the expected amount of DBP/Vitamin D Binding Protein in samples.
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| Linearity | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of DBP/Vitamin D Binding Protein 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 GC / Vitamin D Binding protein ELISA Kit Protocol
The below protocol is a sample protocol for Human GC / Vitamin D Binding protein 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 GC / Vitamin D Binding protein 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 GC / Vitamin D Binding protein 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 GC / Vitamin D Binding protein 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 GC / Vitamin D Binding protein ELISA Kit Protein Information
| UniProt Protein Function: | DBP: This transcriptional activator recognizes and binds to the sequence 5'-RTTAYGTAAY-3' found in the promoter of genes such as albumin, CYP2A4 and CYP2A5. It is not essential for circadian rhythm generation, but modulates important clock output genes. May be a direct target for regulation by the circadian pacemaker component clock. May affect circadian period and sleep regulation. Belongs to the bZIP family. PAR subfamily. |
| UniProt Protein Details: | Protein type:Transcription factor; DNA-binding Chromosomal Location of Human Ortholog: 19q13.3 Biological Process: positive regulation of transcription from RNA polymerase II promoter; regulation of transcription from RNA polymerase II promoter |
| NCBI Summary: | The protein encoded by this gene is a member of the PAR bZIP transcription factor family and binds to specific sequences in the promoters of several genes, such as albumin, CYP2A4, and CYP2A5. The encoded protein can bind DNA as a homo- or heterodimer and is involved in the regulation of some circadian rhythm genes. [provided by RefSeq, Jul 2014] |
| UniProt Code: | Q10586 |
| NCBI GenInfo Identifier: | 1706312 |
| NCBI Gene ID: | 1628 |
| NCBI Accession: | Q10586.1 |
| UniProt Secondary Accession: | Q10586,A2I2P4, |
| UniProt Related Accession: | Q10586 |
| Molecular Weight: | 34,349 Da |
| NCBI Full Name: | D site-binding protein |
| NCBI Synonym Full Names: | D-box binding PAR bZIP transcription factor |
| NCBI Official Symbol: | DBP |
| NCBI Official Synonym Symbols: | DABP |
| NCBI Protein Information: | D site-binding protein |
| UniProt Protein Name: | D site-binding protein |
| UniProt Synonym Protein Names: | Albumin D box-binding protein; Albumin D-element-binding protein; Tax-responsive enhancer element-binding protein 302; TaxREB302 |
| Protein Family: | D site-binding protein |
| UniProt Gene Name: | DBP |
| UniProt Entry Name: | DBP_HUMAN |
