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Typical IκB Standard Curve 100 μL of progressive 2-fold dilutions of the IκB standard included in the kit and run as described ...
IκB STAR ELISA Kit
Description:
IκB STAR ELISA Kit
Trade Name:
Chemicon (Millipore)
Qty/Pk:
96 assays
Background Information:
Test Principle
The UPSTATE® colorimetric STAR (Signal Transduction Assay Reaction) ELISA kit is a solid phase sandwich enzyme linked immunosorbent assay that provides a fast, sensitive method to detect specific levels of signaling targets in whole cell extracts. The IkB plate is coated with a specific mouse monoclonal IkB capture antibody on the microwells of the 96-well clear plate. Sample lysate or the standard included in the kit are incubated in the microwells allowing IkB antigen to be captured in the plate wells. The plate is then washed to remove any non-bound unspecific material. The wells are then incubated with a specific rabbit anti-IkB antibody to detect the capture IkB on the plate well. The unbound detection antibody is washed away followed by incubation with an HRP-conjugated anti-rabbit antibody. This allows for a sensitive enzymatic detection of the sample. After the addition of TMB substrate and stop solution the absorbance is measured at 450 nm using a plate reader.
The entire assay takes less than 5 hours to complete with minimal hands-on time. Many of the reagents are supplied in ready-to use formulations for ease of use. The kit also includes a standard that is run as both a positive control and to develop a standard line of detection.
IkB Background
IKK beta (IkB Kinase, IKKb, I-Kappa-B kinase-beta) is a member of the IKK complex which is composed of IKK alpha, IKK beta, IKK gamma and IKAP. IkB Kinase (IKK) complex is a critical component in NFB regulation. In a majority of unstimulated cells, the NFκB transcription factors exist in their inactive form and are retained in the cytoplasm by the bound inhibitory IkB proteins. For NFB to become activated, it must first disassociate from the inhibitor IκB. This occurs via the serine phosphorylation of IkB on Ser32 and Ser36. Post receptor stimulation, most notably by TNFα, NIK, a serine/threonine kinase, becomes activates and phosphorylates IKKα and IKKβ. Phosphorylation of 2 sites at the activation loop of IKK beta is essential for activation of IKK by TNF and IL1. Once activated, IKK beta autophosphorylates, which in turn decreases IKK activity and prevents prolonged activation of the inflammatory response. Additionally, IKK beta activity can also be regulated by MEKK-1. IKK then binds to and phosphorylates IkBα on the two serine residues. This phosphorylation marks IkB for ubiquination and destruction by the proteosome. This free the NFkB complex and allows it to translocates to the nucleus.
The UPSTATE® colorimetric STAR (Signal Transduction Assay Reaction) ELISA kit is a solid phase sandwich enzyme linked immunosorbent assay that provides a fast, sensitive method to detect specific levels of signaling targets in whole cell extracts. The IkB plate is coated with a specific mouse monoclonal IkB capture antibody on the microwells of the 96-well clear plate. Sample lysate or the standard included in the kit are incubated in the microwells allowing IkB antigen to be captured in the plate wells. The plate is then washed to remove any non-bound unspecific material. The wells are then incubated with a specific rabbit anti-IkB antibody to detect the capture IkB on the plate well. The unbound detection antibody is washed away followed by incubation with an HRP-conjugated anti-rabbit antibody. This allows for a sensitive enzymatic detection of the sample. After the addition of TMB substrate and stop solution the absorbance is measured at 450 nm using a plate reader.
The entire assay takes less than 5 hours to complete with minimal hands-on time. Many of the reagents are supplied in ready-to use formulations for ease of use. The kit also includes a standard that is run as both a positive control and to develop a standard line of detection.
IkB Background
IKK beta (IkB Kinase, IKKb, I-Kappa-B kinase-beta) is a member of the IKK complex which is composed of IKK alpha, IKK beta, IKK gamma and IKAP. IkB Kinase (IKK) complex is a critical component in NFB regulation. In a majority of unstimulated cells, the NFκB transcription factors exist in their inactive form and are retained in the cytoplasm by the bound inhibitory IkB proteins. For NFB to become activated, it must first disassociate from the inhibitor IκB. This occurs via the serine phosphorylation of IkB on Ser32 and Ser36. Post receptor stimulation, most notably by TNFα, NIK, a serine/threonine kinase, becomes activates and phosphorylates IKKα and IKKβ. Phosphorylation of 2 sites at the activation loop of IKK beta is essential for activation of IKK by TNF and IL1. Once activated, IKK beta autophosphorylates, which in turn decreases IKK activity and prevents prolonged activation of the inflammatory response. Additionally, IKK beta activity can also be regulated by MEKK-1. IKK then binds to and phosphorylates IkBα on the two serine residues. This phosphorylation marks IkB for ubiquination and destruction by the proteosome. This free the NFkB complex and allows it to translocates to the nucleus.
Key Applications:
ELISA
Species Reactivity:
Human
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Entrez Gene Summary:
NFKB1 (MIM 164011) or NFKB2 (MIM 164012) is bound to REL (MIM 164910), RELA (MIM 164014), or RELB (MIM 604758) to form the NFKB complex. The NFKB complex is inhibited by I-kappa-B proteins (NFKBIA or NFKBIB, MIM 604495), which inactivate NF-kappa-B by trapping it in the cytoplasm. Phosphorylation of serine residues on the I-kappa-B proteins by kinases (IKBKA, MIM 600664, or IKBKB, MIM 603258) marks them for destruction via the ubiquitination pathway, thereby allowing activation of the NF-kappa-B complex. Activated NFKB complex translocates into the nucleus and binds DNA at kappa-B-binding motifs such as 5-prime GGGRNNYYCC 3-prime or 5-prime HGGARNYYCC 3-prime (where H is A, C, or T; R is an A or G purine; and Y is a C or T pyrimidine).[supplied by OMIM]
UniProt Summary:
FUNCTION: SwissProt: P25963 # Inhibits NF-kappa-B by complexing with and trapping it in the cytoplasm. May be involved in regulation of transcriptional responses to NF-kappa-B, including cell adhesion, immune and proinflammatory responses, apoptosis, differentiation and growth. Controlled by sequential serine-phosphorylation, ubiquitination and degradation. Tyrosine-phosphorylation could only lead to dissociation from NF-kappa-B.
SIZE: 317 amino acids; 35609 Da
SUBUNIT: Interacts with p65 (RELA). Interacts with NKIRAS1 and NKIRAS2. Interacts with HBV protein X.
SUBCELLULAR LOCATION: Cytoplasm.
PTM: Phosphorylated; disables inhibition of NF-kappa-B DNA-binding activity. & Ubiquitinated; subsequent to stimulus-dependent phosphorylation on serines, polyubiquitination targets the protein for rapid degradation via the ubiquitin system.
DISEASE: SwissProt: P25963 # Defects in NFKBIA are a cause of autosomal dominant anhidrotic ectodermal dysplasia with immunodeficiency (AD-EDA-ID) [MIM:164008]. Ectodermal dysplasias (EDs) constitute a heterogeneous group of developmental disorders affecting tissues of ectodermal origin. EDs are characterized by abnormal development of two or more ectodermal structures such as hair, teeth, nails and sweat glands, with or without any additional clinical sign. Each combination of clinical features represents a different type of ectodermal dysplasia.
SIMILARITY: SwissProt: P25963 ## Belongs to the NF-kappa-B inhibitor family. & Contains 5 ANK repeats.
SIZE: 317 amino acids; 35609 Da
SUBUNIT: Interacts with p65 (RELA). Interacts with NKIRAS1 and NKIRAS2. Interacts with HBV protein X.
SUBCELLULAR LOCATION: Cytoplasm.
PTM: Phosphorylated; disables inhibition of NF-kappa-B DNA-binding activity. & Ubiquitinated; subsequent to stimulus-dependent phosphorylation on serines, polyubiquitination targets the protein for rapid degradation via the ubiquitin system.
DISEASE: SwissProt: P25963 # Defects in NFKBIA are a cause of autosomal dominant anhidrotic ectodermal dysplasia with immunodeficiency (AD-EDA-ID) [MIM:164008]. Ectodermal dysplasias (EDs) constitute a heterogeneous group of developmental disorders affecting tissues of ectodermal origin. EDs are characterized by abnormal development of two or more ectodermal structures such as hair, teeth, nails and sweat glands, with or without any additional clinical sign. Each combination of clinical features represents a different type of ectodermal dysplasia.
SIMILARITY: SwissProt: P25963 ## Belongs to the NF-kappa-B inhibitor family. & Contains 5 ANK repeats.
Quality Assurance:
Routinely evaluated by ELISA.
Components:
- 1. Capture Plate pre-coated with anti-IκB antibody: (Part No. 17-485A) One pre-coated 96-stripwell immunoplate sealed in a foil pouch.
- 2. Anti- IκB detection antibody: (Part No. 17-485B) One bottle (11 mL) of anti- IκB detection antibody containing sodium azide, ready to use.
- 3. ELISA Diluent: (Part No. 17-485C) One bottle (25 mL) of ELISA Diluent containing sodium azide, ready to use.
- 4. 25X ELISA Wash Buffer: (Part No. 17-485D) One bottle (50 mL) of 25X ELISA Wash Buffer.
- 5. Anti-Rabbit IgG HRP conjugate: (Part No. 17-485E) One vial (125 μL) of 100X anti-rabbit HRP conjugate.
- 6. HRP Diluent: (Part No. 17-485F) One bottle (25 mL) of HRP Diluent.
- 7. TMB Solution: (Part No. 17-485G) One bottle (25 mL) of stabilized tetramethylbenzidine (TMB), ready to use.
- 8. Stop Solution: (Part No. 17-485H) One bottle (25 mL) of stop solution, ready to use.
- 9. IκB Standard: (Part No. 17-485I) Two vials of IκB standard, lyophilized.
- 10. Plate Covers: Two plate covers.
Brand Family:
Upstate
Sensitivity:
Sensitivity: 62.5 pg/mL.
Range of Detection: 62.5 to 4000 pg/mL
Range of Detection: 62.5 to 4000 pg/mL
UniProt Number:
Storage Conditions:
1 year at 4°C from date of shipment
Detection Methods:
Colorimetric
Gene Symbol:
- NFKBIA
- IKB-alpha
- IKBA
- I-kappa-B-alpha
- MAD-3
- MAD3
- IkappaBalpha
- NFKBI
Product Name:
IκB STAR ELISA Kit
Entrez Gene Number:
Materials Required but Not Delivered:
1. Multi-channel or repeating pipettes
2. Plate shaker (optional)
3. Pipettors & tips capable of accurately measuring 1-1000 μL
4. Graduated serological pipettes
5. 96-well microtiter Plate Reader with 450 nm filter
6. Graphing software for plotting data or graph paper for manual plotting of data
7. Microfuge tubes for standard and sample dilutions
8. Mechanical vortex
9. 1 liter container
10. Distilled or deionized water
2. Plate shaker (optional)
3. Pipettors & tips capable of accurately measuring 1-1000 μL
4. Graduated serological pipettes
5. 96-well microtiter Plate Reader with 450 nm filter
6. Graphing software for plotting data or graph paper for manual plotting of data
7. Microfuge tubes for standard and sample dilutions
8. Mechanical vortex
9. 1 liter container
10. Distilled or deionized water
Analytes Available:
IκB