Protein Name & Synonyms:
Nuclear factor NF-kappa-B p100 subunit (DNA-binding factor KBF2) (H2TF1) (Lymphocyte translocation chromosome 10 protein) (Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2) (Oncogene Lyt-10) (Lyt10) [Cleaved into: Nuclear factor NF-kappa-B p52 subunit]
The olionucleotide/antibody pair provided in this kit recognizes human SOX2 in whole lysates and nuclear extracts.
Number of Targets Detected:
Compatible Sample Types:
- Cell Lysates
- Nuclear Extracts
Method of Detection:
1, 2, or 5 x 96-Well Strip Microplate Kit
The non-canonical NF-κB pathway is an important arm of NF-κB signaling that predominantly functions through activation of the p52/RelB NF-κB complex. This pathway regulates important biological functions, such as lymphoid organogenesis, B-cell survival and maturation, dendritic cell activation, and bone metabolism. Moreover, deregulated non-canonical NF-κB signaling is associated with lymphoid malignancies. In contrast to the canonical NF-κB pathway, in which NF-κB is inactive and retained in the cytoplasm by its inhibitor, IκB, and only transported to the nucleus through modification and degradation of IκB upon stimulation of various factors, the non-canonical pathway depends on the inducible processing of p100, that functions as both the precursor of p52 and a RelB-specific inhibitor. When cells are stimulated by of a subset of TNF receptor family members, the downstream kinase IκB kinase-Ã± (IKKÃ±) is activated and triggers p100 phosphorylation and processing, which generates p52 and results in RelB/p52 NF-κB complex translocation to the nucleus where it regulates the corresponding downstream genes. It is increasingly clear that this pathway of NF-κB activation differs significantly from the canonical NF-κB pathway in its signaling mechanisms. Therefore, better understanding of the mechanism regulating non-canonical NF-κB activation has important therapeutic values.
- Specific transcription factor-DNA binding assay
- Perfect alternative to EMSA
- Easy to perform in an ELISA format
- Non-radioactive assay
- High throughput (96-well plate format)
- Assay can be completed within 5 hours
- 96-well Strip Microplate pre-coated with DNA probes
- DNA Binding Buffer
- Positive Control Sample
- Specific Competitor DNA probe
- Non-specific Competitor DNA probe
- Assay Reagent
- Wash Buffer
- Primary Antibody
- HRP-conjugated Secondary Antibody
- Antibody Diluent Buffer
- TMB One-Step Substrate Reagent
- Stop Solution
Other Materials Required
- Distilled or deionized water
- 100 ml and 1 liter graduated cylinders
- Tubes to prepare sample dilutions
- Absorbent paper
- Precision pipettes to deliver 2 µl to 1 ml volumes
- Adjustable 1-25 ml pipettes for reagent preparation
- Microplate reader capable of measuring absorbance at 450 nm
- Prepare all reagents and samples as instructed in the manual.
- Add 100 µl of sample or positive control to each well.
- Incubate 2 h at RT or O/N at 4 °C.
- Add 100 µl of prepared primary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of prepared HRP-secondary antibody to each well.
- Incubate 1 h at RT.
- Add 100 µl of TMB One-Step Substrate Reagent to each well.
- Incubate 30 min at RT.
- Add 50 µl of Stop Solution to each well.
- Read at 450 nm immediately.
Transcription factor assay of NF-κB p52 from nuclear extracts of HL60 cells or HeLa cells with the RayBio®NF-κBp52 TF Activity Assay.
Transcription factor assay of NF-κB p52 from nuclear extracts of HL60 cells or HeLa cells with the specific competitor or non-specific competitor. The result shows specific binding of NF-κB p52 to the NF-κB non-canonical DNA binding site.
Upon receipt, the positive control should be removed and stored at -20° or -80°C. The remainder of the kit can be stored for up to 6 months at 2-8°C from the date of shipment. Opened Microplate Wells or reagents may be stored for up to 1 month at 2° to 8°C. Return unused wells to the pouch containing desiccant pack, reseal along entire edge.
Note: The kit can be used within one year if the whole kit is stored at -20°C upon receipt. Avoid repeated freeze-thaw cycles.