Rat NFқb-p65 ELISA Kit

Catalog No.: BYEK3040

Size: 96T

Range: 100 ng/L - 1200 ng/L

Sensitivity: 10 ng/L

Storage and Expiration: Store at 2-8℃ for 6 months.

Application: For quantitative detection of NFқb-p65 in rat serum, plasma, tissue homogenate or cell culture supernatant.

Introduction

NF-kappa-B is a pleiotropic transcription factor present in almost all cell types and is the endpoint of a series of signal transduction events that are initiated by a vast array of stimuli related to many biological processes such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. NF-kappa-B is a homo- or heterodimeric complex formed by the Rel-like domain-containing proteins RELA/p65, RELB, NFKB1/p105, NFKB1/p50, REL and NFKB2/p52 and the heterodimeric p65-p50 complex appears to be most abundant one. In unstimulated cells, NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins. NF-κB-activating agents can induce the phosphorylation of IκB proteins, targeting them for rapid degradation through the ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus where it regulates gene expression.

Principle of the Assay  

This kit was based on standard sandwich enzyme-linked immune-sorbent assay technology. The purified anti- NFқb-p65 antibody was pre-coated onto 96-well plates. And the HRP conjugated anti- NFқb-p65 antibody was used as detection antibodies. The standards, test samples and HRP conjugated detection antibody were added to the wells subsequently, mixed and incubated, then, unbound conjugates were washed away with wash buffer. TMB substrates (A & B) were used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the NFқb-p65 amount of sample captured in plate. Read the O.D. absorbance at 450nm in a microplate reader, and then the concentration of NFқb-p65 can be calculated.

Kit components

1. One 96-well plate pre-coated with anti- rat NFқb-p65 antibody

2. Standard: 0.5ml (1800 ng/L)

3. Standard diluent buffer: 1.5 ml

4. Wash buffer (30×): 20 ml. Dilution: 1:30

5. Sample diluent buffer: 6 ml

6. HRP conjugated anti- rat NFқb-p65 antibody (RTU): 6ml

7. Stop solution: 6 ml

8. TMB substrate A:  6ml

9. TMB substrate B:  6ml

10. Plate sealer:   2

11. Hermetic bag:  1

Material Required But Not Provided

1. 37℃ incubator
2. Microplate reader (wavelength: 450nm)
3. Precise pipette and disposable pipette tips
4. Automated plate washer
5. ELISA shaker
6. 1.5ml of Eppendorf tubes
7. Absorbent filter papers
8. Plastic or glass container with volume of above 1L