Human CACYBP (Calcyclin Binding Protein) ELISA Kit (HUES01785)



ELISA Kit Technical ManualMSDS

Human CACYBP (Calcyclin Binding Protein) ELISA Kit

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 CACYBP . 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 CACYBP 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 CACYBP, 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 CACYBP. The concentration of Human CACYBP in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Assay time4.5h
Detection MethodColormetric
Detection Range78.13-5000 pg/mL
Sensitivity46.88 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids


This kit recognizes Human CACYBP in samples. No significant cross-reactivity or interference between Human CACYBP and analogues was observed.

Typical Data

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.

O.D Average Corrected


Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CACYBP were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CACYBP were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Mean (pg/mL)267.10545.071922.90266.03596.921803.39
Standard deviation13.8632.4385.5713.7526.1581.69
C V (%)5.195.954.455.174.384.53


The recovery of Human CACYBP 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)95-109101
EDTA plasma (n=5)90-10396
Cell culture media (n=5)90-10495


Samples were spiked with high concentrations of Human CACYBP 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)
1:2Range (%)94-10791-10389-102
Average (%)999795
1:4Range (%)90-10483-9783-99
Average (%)988890
1:8Range (%)87-10184-9586-99
Average (%)928991
1:16Range (%)91-10781-9488-100
Average (%)988794

Kit Components & Storage

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 X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human CACYBP (Calcyclin Binding Protein) ELISA Kit (HUES01785) Assay procedure

    1. 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.
    2. Aliquot 100 µL of standard solutions into the standard wells.
    3. Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100 µL of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37 °C.
    6. 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.
    7. 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.
    8. Add 100 µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37 °C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. 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.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human CACYBP (Calcyclin Binding Protein) ELISA Kit (HUES01785) Protein Information

UniProt Protein Function:CACYBP: a nuclear/cytoplasmic protein involved in calcium-dependent ubiquitination and proteosomal degradation of target proteins. A possible molecular bridge in ubiquitin E3 complexes. Participates in the ubiquitin-mediated degradation of beta-catenin (CTNNB1). Interacts with proteins of the S100 family at physiological calcium concentrations. Component of some large E3 complexes and interacts directly with SIAH1, SIAH2 and SKP1A. Cytoplasmic at low calcium concentrations. In neuroblastoma cells, after a retinoic acid (RA) induction and calcium increase, it localizes in both the nucleus and cytoplasm. The nuclear fraction may be phosphorylated. Two alternatively spliced isoforms have been described.
UniProt Protein Details:

Protein type:Adaptor/scaffold; Ubiquitin conjugating system

Chromosomal Location of Human Ortholog: 1q24-q25

Cellular Component: nucleoplasm; neuron projection; nuclear envelope lumen; cytoplasm; beta-catenin destruction complex

Molecular Function:protein binding; protein homodimerization activity

Biological Process: positive regulation of DNA replication; cardiac muscle cell differentiation; aging

NCBI Summary:The protein encoded by this gene is a calcyclin binding protein. It may be involved in calcium-dependent ubiquitination and subsequent proteosomal degradation of target proteins. It probably serves as a molecular bridge in ubiquitin E3 complexes and participates in the ubiquitin-mediated degradation of beta-catenin. Two alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:Q9HB71
NCBI GenInfo Identifier:46576651
NCBI Gene ID:27101
NCBI Accession:Q9HB71.2
UniProt Secondary Accession:Q9HB71,O60666, Q5R370, Q5R371, B2ZWH2, B3KSF1,
UniProt Related Accession:Q9HB71
Molecular Weight:21,228 Da
NCBI Full Name:Calcyclin-binding protein
NCBI Synonym Full Names:calcyclin binding protein
NCBI Official Symbol:CACYBP
NCBI Official Synonym Symbols:SIP; GIG5; PNAS-107; S100A6BP
NCBI Protein Information:calcyclin-binding protein; hCacyBP; S100A6-binding protein; Siah-interacting protein (SIP); growth-inhibiting gene 5 protein
UniProt Protein Name:Calcyclin-binding protein
UniProt Synonym Protein Names:S100A6-binding protein; Siah-interacting protein
Protein Family:Calcyclin-binding protein
UniProt Gene Name:CACYBP
UniProt Entry Name:CYBP_HUMAN
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