Human CREB (Cyclic AMP Response Element Binding Protein) ELISA Kit (HUES01981)
- SKU:
- HUES01981
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P16220
- Sensitivity:
- 0.19ng/mL
- Range:
- 0.31-20ng/mL
- ELISA Type:
- Sandwich
- Reactivity:
- Human
- Tested Sample Types:
- Serum, plasma and other biological fluids
Description
| Assay type: | Sandwich |
| Format: | 96T |
| Assay time: | 4.5h |
| Reactivity: | Human |
| Detection Method: | Colormetric |
| Detection Range: | 0.31-20 ng/mL |
| Sensitivity: | 0.19 ng/mL |
| Sample Volume Required Per Well: | 100µL |
| Sample Type: | Serum, plasma and other biological fluids |
| Specificity: | This kit recognizes Human CREB in samples. No significant cross-reactivity or interference between Human CREB and analogues was observed. |
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human CREB. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human CREB and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human CREB, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human CREB. The concentration of Human CREB in samples can be calculated by comparing the OD of the samples to the standard curve.
| UniProt Protein Function: | Function: This protein binds the cAMP response element (CRE), a sequence present in many viral and cellular promoters. CREB stimulates transcription on binding to the CRE. Transcription activation is enhanced by the TORC coactivators which act independently of Ser-133 phosphorylation. Implicated in synchronization of circadian rhythmicity. |
| UniProt Protein Details: | Subunit structure: Interacts with PPRC1. Binds DNA as a dimer. This dimer is stabilized by magnesium ions. Interacts, through the bZIP domain, with the coactivators TORC1/CRTC1, TORC2/CRTC2 and TORC3/CRTC3. When phosphorylated on Ser-133, binds CREBBP By similarity. Interacts (phosporylated form) with TOX3. Interacts with ARRB1. Ref. 8 Ref. 9 Ref. 11 Ref. 13 Ref. 14 Ref. 15 Ref. 16 Ref. 20 Subcellular location: Nucleus Ref. 12. Post-translational modification: Stimulated by phosphorylation. Phosphorylation of both Ser-133 and Ser-142 in the SCN regulates the activity of CREB and participates in circadian rhythm generation. Phosphorylation of Ser-133 allows CREBBP binding By similarity. Phosphorylated upon DNA damage, probably by ATM or ATR. Ref. 10 Ref. 17 Ref. 19Sumoylated by SUMO1. Sumoylation on Lys-304, but not on Lys-285, is required for nuclear localization of this protein. Sumoylation is enhanced under hypoxia, promoting nuclear localization and stabilization. Ref. 12 Involvement in Disease: Defects in CREB1 may be a cause of angiomatoid fibrous histiocytoma (AFH) [ MIM:612160]. A distinct variant of malignant fibrous histiocytoma that typically occurs in children and adolescents and is manifest by nodular subcutaneous growth. Characteristic microscopic features include lobulated sheets of histiocyte-like cells intimately associated with areas of hemorrhage and cystic pseudovascular spaces, as well as a striking cuffing of inflammatory cells, mimicking a lymph node metastasis. Note=A chromosomal aberration involving CREB1 is found in a patient with angiomatoid fibrous histiocytoma. Translocation t(2;22)(q33;q12) with CREB1 generates a EWSR1/CREB1 fusion gene that is most common genetic abnormality in this tumor type. Sequence similarities: Belongs to the bZIP family. Contains 1 bZIP domain. Contains 1 KID (kinase-inducible) domain. |
| NCBI Summary: | This gene encodes a transcription factor that is a member of the leucine zipper family of DNA binding proteins. This protein binds as a homodimer to the cAMP-responsive element, an octameric palindrome. The protein is phosphorylated by several protein kinases, and induces transcription of genes in response to hormonal stimulation of the cAMP pathway. Alternate splicing of this gene results in two transcript variants encoding different isoforms. [provided by RefSeq] |
| UniProt Code: | P16220 |
| NCBI GenInfo Identifier: | 117434 |
| NCBI Gene ID: | 1385 |
| NCBI Accession: | P16220. 2 |
| UniProt Secondary Accession: | P16220,P21934, Q9UMA7, |
| UniProt Related Accession: | P16220,Q16366,Q16367,Q4ZG78,Q53RU9,Q53X93,Q5U0J5,Q6V963 |
| Molecular Weight: | 40 kDa |
| NCBI Full Name: | Cyclic AMP-responsive element-binding protein 1 |
| NCBI Synonym Full Names: | cAMP responsive element binding protein 1 |
| NCBI Official Symbol: | CREB1 |
| NCBI Official Synonym Symbols: | CREB; MGC9284 |
| NCBI Protein Information: | cyclic AMP-responsive element-binding protein 1; CREB-1; OTTHUMP00000163864; OTTHUMP00000163865; OTTHUMP00000206660; OTTHUMP00000206662; OTTHUMP00000206667; transactivator protein; active transcription factor CREB; cAMP-response element-binding protein-1; cAMP-responsive element-binding protein 1 |
| UniProt Protein Name: | Cyclic AMP-responsive element-binding protein 1 |
| Protein Family: | CREB-binding protein |
| UniProt Gene Name: | CREB1 |
| UniProt Entry Name: | CREB1_HUMAN |
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
| Concentration (ng/mL) | O.D | Average | Corrected |
| 20 | 2.372 2.408 | 2.39 | 2.31 |
| 10 | 1.612 1.652 | 1.632 | 1.552 |
| 5 | 0.891 0.859 | 0.875 | 0.795 |
| 2.5 | 0.46 0.486 | 0.473 | 0.393 |
| 1.25 | 0.254 0.228 | 0.241 | 0.161 |
| 0.63 | 0.192 0.164 | 0.178 | 0.098 |
| 0.31 | 0.12 0.14 | 0.13 | 0.05 |
| 0 | 0.08 0.08 | 0.08 | -- |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CREB were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CREB were tested on 3 different plates, 20 replicates in each plate.
| Intra-assay Precision | Inter-assay Precision | |||||
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 20 | 20 | 20 |
| Mean (ng/mL) | 0.98 | 1.73 | 8.77 | 1.08 | 1.87 | 8.64 |
| Standard deviation | 0.06 | 0.08 | 0.46 | 0.07 | 0.10 | 0.41 |
| C V (%) | 6.12 | 4.62 | 5.25 | 6.48 | 5.35 | 4.75 |
Recovery
The recovery of Human CREB spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
| Sample Type | Range (%) | Average Recovery (%) |
| Serum (n=5) | 93-105 | 100 |
| EDTA plasma (n=5) | 92-105 | 99 |
| Cell culture media (n=5) | 86-97 | 91 |
Linearity
Samples were spiked with high concentrations of Human CREB and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
| Serum (n=5) | EDTA plasma (n=5) | Cell culture media (n=5) | ||
| 1:2 | Range (%) | 99-114 | 87-99 | 87-101 |
| Average (%) | 105 | 93 | 94 | |
| 1:4 | Range (%) | 92-104 | 84-96 | 86-99 |
| Average (%) | 98 | 91 | 93 | |
| 1:8 | Range (%) | 91-107 | 87-99 | 85-96 |
| Average (%) | 97 | 92 | 90 | |
| 1:16 | Range (%) | 95-106 | 82-93 | 85-100 |
| Average (%) | 100 | 87 | 92 |
An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
| Item | Specifications | Storage |
| Micro ELISA Plate(Dismountable) | 8 wells ×12 strips | -20°C, 6 months |
| Reference Standard | 2 vials | |
| Concentrated Biotinylated Detection Ab (100×) | 1 vial, 120 µL | |
| Concentrated HRP Conjugate (100×) | 1 vial, 120 µL | -20°C(shading light), 6 months |
| Reference Standard & Sample Diluent | 1 vial, 20 mL | 4°C, 6 months |
| Biotinylated Detection Ab Diluent | 1 vial, 14 mL | |
| HRP Conjugate Diluent | 1 vial, 14 mL | |
| Concentrated Wash Buffer (25×) | 1 vial, 30 mL | |
| Substrate Reagent | 1 vial, 10 mL | 4°C(shading light) |
| Stop Solution | 1 vial, 10 mL | 4°C |
| Plate Sealer | 5 pieces | |
| Product Description | 1 copy | |
| Certificate of Analysis | 1 copy |
- Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- Aliquot 100 µL of standard solutions into the standard wells.
- Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
- Add 100 µL of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37 °C.
- Aspirate the liquid from each well, do not wash. Immediately add 100 µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37 °C.
- Aspirate or decant the solution from the plate and add 350 µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- Add 100 µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37 °C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- Add 90 µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37 °C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.
