Description

Nori Human 14-3-3 eta ELISA Kit Summary

Alternative names for 14-3-3 eta: YWHAH, YWHA1

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background:

14-3-3 proteins are a family of conserved regulatory molecules that are expressed in all eukaryotic cells. 14-3-3 proteins have the ability to bind a multitude of functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors. More than 200 signaling proteins have been reported as 14-3-3 ligands. The 14-3-3 proteins eluted in the 14th fraction of bovine brain homogenate and were found on positions 3.3 of subsequent electrophoresis by Moore and Perez (1967). Elevated amounts of 14-3-3 proteins are found in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease.^[1] There are seven genes that encode seven distinct 14-3-3 proteins in most mammals. Eukaryotes can tolerate the loss of a single 14-3-3 gene if multiple genes are expressed, however deletion of all 14-3-3s (as experimentally determined in yeast) results in death. 14-3-3 proteins can be considered evolved members of the Tetratrico Peptide Repeat (TPR) superfamily, generally have 9 or 10 alpha helices, and usually form homo- and/or hetero-dimer interactions along their amino-termini helices. These proteins contain a number of known common modification domains, including regions for divalent cation interaction, phosphorylation^[2] & acetylation, and proteolytic cleavage, among others established and predicted. 14-3-3 proteins play an isoform-specific role in class switch recombination. They are believed to interact with the protein Activation-Induced (Cytidine) Deaminase in mediating class switch recombination. Phosphorylation of Cdc25C by CDS1 and CHK1 creates a binding site for the 14-3-3 family of phosphoserine binding proteins. Binding of 14-3-3 has little effect on Cdc25C activity, and it is believed that 14-3-3 regulates Cdc25C by sequestering it to the cytoplasm, thereby preventing the interactions with CycB-Cdk1 that are localized to the nucleus at the G2/M transition.^[3]

References

1. Takahashi H et al. (1999). Clinical and Diagnostic Laboratory Immunology 6 (6): 983–5.
2. Madhurima S et al. (2012). Biochemical Journal. doi:10.1042/BJ20120938.
3. Jahn TP et al. (2002). The Journal of Biological Chemistry 277 (8): 6353–6358.

Product Citations