Human Nuclear Factor Kappa B / NkB ELISA Kit

SKU:
HUFI02661
€299

Description

ELISA Kit Technical ManualMSDS

Human NFkappaB (Nuclear Factor Kappa B) ELISA Kit - Information

The Assay Genie Human NFkappaB (Nuclear Factor Kappa B) ELISA Kit can assay for Human NFkappaB in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How do our Human NFkappaB (Nuclear Factor Kappa B) ELISA Kits Work?

The Assay Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, Assay Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At Assay Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound Human NFkappaB is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Human NFkappaB (Nuclear Factor Kappa B) ELISA Kit Data

Product Code

HUFI02661

Uniprot P19838
Alias

NFkappaB, Nuclear Factor Kappa B

Detection method

Sandwich ELISA, Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of Human NFkappaB concentrations in serum plasma and other biological fluids.

Size

96T

Range

0.313-20ng/ml

Sensitivity

0.188ng/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of Human NFkappaB and the recovery rates were calculated by comparing the measured value to the expected amount of Human NFkappaB in samples.

MatrixRecovery range(%)Average(%)
serum(n=5) 88-105 99
EDTA plasma(n=5) 88-99 92
UFH plasma(n=5) 90-99 94
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human NFkappaB and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample1:21:41:8
serum(n=5) 89-103% 94-105% 87-105%
EDTA plasma(n=5) 82-94% 90-99% 88-101%
UFH plasma(n=5) 83-99% 82-99% 87-96%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Human NFkappaB (Nuclear Factor Kappa B) ELISA Kit Protocol

The below protocol is a sample protocol for Human NFkappaB (Nuclear Factor Kappa B) ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISA Kits allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human NFkappaB Antibody present in their sample.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Assay Protocol:

1. Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2. Aliquot 0.1ml standard solutions into the standard wells.
3. Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4. Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5. Seal the plate with a cover and incubate at 37 °C for 90 min.
6. Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7. Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8. Seal the plate with a cover and incubate at 37°C for 60 min.
9. Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10. Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11. Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12. Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13. Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

Human NFkappaB (Nuclear Factor Kappa B) ELISA Kit components

96 Assays

Storage

ELISA Microplate (Dismountable) 8×12 strips 4°C for 6 months
Lyophilized Standard 2 4°C/-20°C
Sample/Standard Dilution Buffer 20ml 4°C
Biotin-labeled Antibody(Concentrated) 120ul 4°C (Protect from light)
Antibody Dilution Buffer 10ml 4°C
HRP-Streptavidin Conjugate(SABC) 120ul 4°C (Protect from light)
SABC Dilution Buffer 10ml 4°C
TMB Substrate 10ml 4°C (Protect from light)
Stop Solution 10ml 4°C
Wash Buffer(25X) 30ml 4°C
Plate Sealer 5 -

Other materials and equipment required:

The Assay Genie Human NFkappaB (Nuclear Factor Kappa B) ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir

Sample Preparation

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type Protocol

Serum

If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

Plasma

Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.

Urine & Cerebrospinal Fluid

Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.

Cell culture supernatant

Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.

Cell lysates

Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Tissue homogenates

The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.

Tissue lysates

Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.

Breast Milk

Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

Human NFkappaB Protein Information

UniProt Protein Function: NFkB-p105: a transcription factor of the nuclear factor-kappaB ( NFkB) group. Undergoes cotranslational processing by the 26S proteasome to produce a 50 kD protein. The 105 kD protein is a Rel protein-specific transcription inhibitor and the 50 kD protein is a DNA binding subunit of NFkB. NFkB is a transcription regulator that is activated by various intra- and extra-cellular stimuli such as cytokines, oxidant-free radicals, ultraviolet irradiation, and bacterial or viral products. Activated NFkB translocates into the nucleus and stimulates the expression of genes involved in a wide variety of biological functions. Inappropriate activation of NFkB has been associated with a number of inflammatory diseases while persistent inhibition of NFkB leads to inappropriate immune cell development or delayed cell growth. There are five NFkB proteins in mammals (RelA/NFkB-p65, RelB, c-Rel, NF-_B1/NFkB-p105, and NF-_B2/NFkB-p100). They form a variety of homodimers and heterodimers, each of which activates its own characteristic set of genes. Two alternatively spliced isoforms have been described.
UniProt Protein Details:

Protein type:DNA-binding; Transcription factor

Chromosomal Location of Human Ortholog: 4q24

Cellular Component: cytoplasm; cytosol; mitochondrion; neuron projection; nucleoplasm; nucleus

Molecular Function:actinin binding; chromatin binding; heat shock protein binding; identical protein binding; protein binding; protein heterodimerization activity; protein homodimerization activity; transcription factor activity; transcription factor binding

Biological Process: activation of NF-kappaB transcription factor; apoptosis; I-kappaB kinase/NF-kappaB cascade; inflammatory response; innate immune response; membrane protein intracellular domain proteolysis; MyD88-dependent toll-like receptor signaling pathway; MyD88-independent toll-like receptor signaling pathway; negative regulation of apoptosis; negative regulation of cellular protein metabolic process; negative regulation of cholesterol transport; negative regulation of inflammatory response; negative regulation of interleukin-12 biosynthetic process; negative regulation of transcription from RNA polymerase II promoter; nerve growth factor receptor signaling pathway; positive regulation of interferon type I production; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; response to copper ion; response to oxidative stress; stimulatory C-type lectin receptor signaling pathway; stress-activated MAPK cascade; T cell receptor signaling pathway; toll-like receptor 10 signaling pathway; toll-like receptor 2 signaling pathway; toll-like receptor 3 signaling pathway; toll-like receptor 4 signaling pathway; toll-like receptor 5 signaling pathway; toll-like receptor 9 signaling pathway; toll-like receptor signaling pathway; transcription from RNA polymerase II promoter

Disease: Immunodeficiency, Common Variable, 12

NCBI Summary: This gene encodes a 105 kD protein which can undergo cotranslational processing by the 26S proteasome to produce a 50 kD protein. The 105 kD protein is a Rel protein-specific transcription inhibitor and the 50 kD protein is a DNA binding subunit of the NF-kappa-B (NFKB) protein complex. NFKB is a transcription regulator that is activated by various intra- and extra-cellular stimuli such as cytokines, oxidant-free radicals, ultraviolet irradiation, and bacterial or viral products. Activated NFKB translocates into the nucleus and stimulates the expression of genes involved in a wide variety of biological functions. Inappropriate activation of NFKB has been associated with a number of inflammatory diseases while persistent inhibition of NFKB leads to inappropriate immune cell development or delayed cell growth. Alternative splicing results in multiple transcript variants encoding different isoforms, at least one of which is proteolytically processed. [provided by RefSeq, Feb 2016]
UniProt Code: P19838
NCBI GenInfo Identifier: 21542418
NCBI Gene ID: 4790
NCBI Accession: P19838.2
UniProt Secondary Accession: P19838,Q68D84, Q86V43, Q8N4X7, Q9NZC0, A8K5Y5, B3KVE8
UniProt Related Accession: P19838
Molecular Weight: 85,520 Da
NCBI Full Name: Nuclear factor NF-kappa-B p105 subunit
NCBI Synonym Full Names: nuclear factor of kappa light polypeptide gene enhancer in B-cells 1
NCBI Official Symbol: NFKB1
NCBI Official Synonym Symbols: p50; KBF1; p105; EBP-1; CVID12; NF-kB1; NFKB-p50; NFkappaB; NF-kappaB; NFKB-p105; NF-kappa-B
NCBI Protein Information: nuclear factor NF-kappa-B p105 subunit
UniProt Protein Name: Nuclear factor NF-kappa-B p105 subunit
UniProt Synonym Protein Names: DNA-binding factor KBF1; EBP-1; Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1
UniProt Gene Name: NFKB1
UniProt Entry Name: NFKB1_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
Research Area:
Cell Death
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