Human, Mouse, Rat RelA/NFkB p65 ELISA Kit (Colorimetric) Summary
| Description |
APPROPRIATE CONTROLS TO INCLUDE:
Following is a list of suggested controls to include with each analysis:
1. No capture antibody added to well.
2. No lysate added to well.
3. No capture antibody or lysate added to well.
4. Positive control: use a cell line or tissue known to constitutively express p65 or a recombinantly expressed p65.
5. Negative control: use a cell line or tissue known to not express p65.
Please note that this kit has not yet been tested or used with any of the reporter cell lines and is therefore not guaranteed for detection of the target protein these samples. Contains reagents and protocol to prepare whole, nuclear, and cytoplasmic cell fractions. Multiple samples can be analyzed in a low-volume, high-throughput format. Full analysis complete in just hours. Allows direct measurement of changes in p65 translocation. Allows study of NF-kB activation without gel-shift assay. [This product is the same as Imgenex's NF-kappaB p65 ActivELISA Kit, IMK-503]. |
| Specificity |
For the detection of cytoplasmic, nuclear and total free NF-kB/p65 in the nucleus of either cells or tissues. Human, mouse, rat and sheep reported in the literature. |
| Standard Curve Range |
15.6 - 1000 ng/ml |
| Sensitivity |
15.6 ng/ml |
| Assay Type |
Sandwich ELISA |
| Sample Volume |
0.1 ml |
| Kit Type |
ELISA Kit (Colorimetric) |
| Gene |
RELA |
Applications/Dilutions
| Dilutions |
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| Publications |
Read Publications using NBP2-29661 in the following applications:
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Packaging, Storage & Formulations
| Storage |
Storage is content dependent. |
Kit Components
|
Components
|
- 1 M DTT (for Nuclear Extraction from tissue) 100 ul
- 10 mM DTT (for Nuclear Lysis Buffer) 500 ul
- 10% Detergent Solution 10 ml
- 100 mM PMSF 10 ml
- 100X Protease Inhibitor Cocktail (PIC) 100 ul
- 10X Hypotonic Lysis Buffer 10 ml
- 10X PBS 2 x 50 ml
- 1X Nuclear Extraction Buffer 10 ml
- AKP-Conjugated Secondary Ab 10 ul
- BSA 2 x 0.5 g
- Capture Antibody 200ul
- Coating Buffer 2 x 10 ml
- Detecting Antibody 200 ul
- ELISA Plates 2
- Manual 1
- pNPP 4 x 5 mg
- pNPP Substrate Buffer 2 x 10 ml
- Recombinant p65 Standard 2 vials lyophilized(0.42 ug/vial)
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Alternate Names for Human, Mouse, Rat RelA/NFkB p65 ELISA Kit (Colorimetric)
Background
Activation of the NF-kB pathway can be triggered by many factors including TNFalpha, UV, IL-1, lipopolysaccharide (LPS), mitogens, and phorbol esters. NF-kB is controlled by a family of inhibitory proteins called, IkBs. IkB proteins are phosphorylated by IkB kinase complex consisting of at least three proteins, IKK1/alpha, IKK2/Beta, and IKK3/gamma. External stimuli such as tumor necrosis factor or other cytokines initiate a signal transduction cascade that leads to the activation of IkB-kinase complex, which then specifically phosphorylates IkBalpha on Serine-32 and Serine-36. Phosphorylation of these sites leads to ubiquitination of IkBalpha and subsequent degradation by the 26 S proteasome. Degradation of IkBalpha results in unmasking of the nuclear localization signal of NF-kB dimers, which subsequently translocates to the nucleus and acts as a transcription factor for genes controlling inflammatory cytokines, adhesion molecules, and other proteins. Thus the nuclear levels of p65 may correlate positively with the activation of NF-kB pathway.
Limitations
This product is for research use only and is not approved for use in humans or in clinical diagnosis. ELISA Kits are
guaranteed for 6 months from date of receipt.
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Product General Protocols
View specific protocols for RelA/NFkB p65 ELISA Kit (NBP2-29661):
Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.
FAQs for RelA/NFkB p65 ELISA Kit (NBP2-29661). (Showing 1 - 3 of 3 FAQs).
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Can we perform this ELISA on the frozen brain tissues or every time you need fresh tissue?
- We have not evaluated this kit on brain tissue, neither fresh nor frozen. The the kit is quality controlled using the recombinant protein standard in the kit. However, the kit has been published using different types of tissues. Please follow this link for representative publications, including brain tissue NBP2-29661. Ultimately, the researcher will need to empirically determine if the kit is suitable for their brain tissue and how the results from frozen and fresh brain tissue compare.
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How do I thaw the 100X protease inhibitor cocktail that comes with the NF-kB p65 ELISA kit?
- It is recommended that you leave it at room temp for 30mins, or 4 hours at 4C
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What is the difference between the Quantikine and Duoset ELISA kits?
- Our Quantikine kits are fully optimized and validated for the sample types listed on the product specific webpage and datasheet, as this can vary between kits. Each kit is supplied ready to use with one pre-coated 96-well plate, a detection antibody directly conjugated to HRP, and all other necessary reagents. These kits are ideal for researchers who want the convenience of a ready to use and optimized ELISA product. Some of these kits are also available prepackaged in larger 6 and 50 plate sizes.Our DuoSet Kits, in contrast, are ELISA development kits containing the capture and detection antibody, the mass-value calibrated standard, and streptavidin-HRP to prepare approximately 5 or 15 plates. Ancillary reagents will need to be used/purchased, and for most kits, we will recommend one of our Ancillary Reagent Kits, which contain the reagents we use ourselves in-house. DuoSet kits are validated only for cell culture supernatant samples and therefore require further development and validation for accurate measurement in more complex samples such as serum and plasma. Our DuoSet Kits offer an economical, flexible alternative for the experienced ELISA user.