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ELISA-ONE™ for NF-κB p65

ELISA-ONE for Phospho NF-κB p65 (pS536)

Specificity

ELISA-ONE for phospho NF-κB p65 (pS536) are ready to use ELISA kits for the detection of endogenous levels of NF-κB p65 when phosphorylated at Ser536.

ELISA-ONE for phospho NF-κB p65 (pS536) is comprised of a precoated 96 well (12×8 strips) plate and an antibody pair, recognizing the phospho-Ser536 epitope and a distal epitope of NF-κB p65.

The antibody pair recognizes human and mouse NF-κB p65. However, other species should be tested on a case by case basis.

 

ELISA-ONE for Total NF-κB p65

Specificity

ELISA-ONE for total NF-κB p65 are ready to use ELISA kits for the detection of endogenous levels of NF-κB p65 irrespective of phosphorylation status.

ELISA-ONE for total NF-κB p65 is comprised of a precoated 96 well (12×8 strips) plate and an antibody pair, recognizing two invariants epitopes of NF-κB p65.

The antibody pair recognizes human and mouse NF-κB p65. However, other species should be tested on a case by case basis.

 

ELISA-ONE for Phospho & Total NF-κB p65

Specificity

ELISA-ONE for phospho and total NF-κB p65 are ready to use ELISA kits for the detection of endogenous levels of NF-κB p65 in its phosphorylated and unphosphorylated form.

ELISA-ONE for phospho and total NF-κB p65 is comprised of a precoated 96 well (12×8 strips) plate and two antibody pairs, recognizing the phospho-Ser536 epitope and an invariant epitope of NF-κB p65.

The antibody pair recognizes human and mouse NF-κB p65. However, other species should be tested on a case by case basis.

 

Technical Specifications

GenBank Accessions
NF-κB p65NP_068810

 

Species Cross-reactivity

Tested: Human, mouse

Kit Contents

  • 1x 96 well (12×8 strips) CaptSure assay plate
  • 1x adherent plate seal
  • 1x Lysis Buffer (5X)
  • 1x Enhancer Solution
  • 1x Wash Buffer (10X)
  • 1x Detection Reagent
  • 1x Stop Solution
  • 1x Lyophilized Positive Control Lysate
  • 1x Capture Antibody Reagent 1
  • 1x Capture Antibody Reagent 2 (phospho / total target kits only)
  • 1x Detection Antibody Reagent 1
  • 1x Detection Antibody Reagent 2 (phospho / total target kits only).

 

NF-κB p65 Signaling Pathway

The nuclear factor κB transcription factor family plays a pivotal role in inflammatory and immune responses. There are five family known members, namely p65 (RelA), c‐Rel, RelB, NF‐κB1 (p105/p50) and NF‐κB2 (p100/p52). The p50 and p52 products form dimeric complexes with Rel proteins, which bind DNA and regulate transcription. The most abundant form in many cell types is the p65/p50 complex. In unstimulated cells, NF‐κB is inactive in the cytoplasm, bound by I‐κB proteins. Activation of the NF‐κB pathway induces phosphorylation of I‐κBs, targeting them for degradation through the ubiquitin proteasome pathway. Free NF‐κB can then enter the nucleus, where it regulates expression of its target genes.

DIAGRAM_ELISA-ONE_NF-kB pathway

 

NF-κB p65 References

Buss, H., Handschick, K., Jurrmann, N., Pekkonen, P., Beuerlein, K., Müller, H., Wait, R., Saklatvala, J., Ojala, PM., Schmitz, ML., Naumann, M., Kracht. M. Cyclin-dependent kinase 6 phosphorylates NF-κB P65 at serine 536 and contributes to the regulation of inflammatory gene expression. PLoS One. 2012;7(12):e51847. doi: 10.1371/journal.pone.0051847. Epub 2012 Dec 26.

Pan, X., Jiang, B., Liu, J., Ding, J., Li, Y., Sun, R., Peng, L., Qin, C., Fang, S., Li, G. STC1 promotes cell apoptosis via NF-κB phospho-P65 Ser536in cervical cancer cells. Oncotarget. 2017 Jul 11;8(28):46249–46261. doi: 10.18632/oncotarget.17641.

Zhang, L., Shao, L., Creighton, CJ., Zhang, Y., Xin, L., Ittmann, M., Wang, J. Function of phosphorylation of NF-kB p65 ser536 in prostate cancer oncogenesis. Oncotarget. 2015 Mar 20;6(8):6281–4.

 

Can’t find the target you are looking for? Please don’t hesitate to get in touch and let us know your requirements.

Phosphorylated and total protein level

Treatment of HeLa cells with TNFα triggers phosphorylation of NF-κB at Ser536 but does not affect the total level of NF-κB (top panel).

 

ELISA-ONE vs Competitors

DATA_ELISA-ONE_vs_Competitors

ELISA-ONE demonstrates a wider dynamic range and lower background compared with commercially available ready to use competitor ELISA kits.

 

Intra & inter plate variabilityDATA_ELISA-ONE_Intra_Iner Plate CV

Mean, standard deviation (SD) and coefficient of variance (%CV) were measured among the same plate (n=24) and four different plates.

 

Frequently Asked Questions (FAQs) for ELISA‐ONE

1. Can I use a different ELISA plate to the one that is supplied?

ELISA‐ONE microplates are specific to ELISA‐ONE and cannot be substituted with any other type of 96 well microplate. ELISA‐ONE microplates, which rely on our CaptSure™ technology, are optimized to provide an active surface for binding the immunocomplex by means of the interaction between the CaptSure antibody precoated onto the plate and the CaptSure peptide conjugated to the capture antibody.

2. How are ELISA-ONE microplates supplied?

ELISA‐ONE microplates are supplied in a 12×8 strip well plate. The plate format allows the user to use only the number of the strip wells needed, without incurring additional waste when performing small scale experiments.

3. How are ELISA-ONE microplates stored?

ELISA‐ONE microplates can be stored at +4°C or room temperature. If stored at +4°C, the strip wells should be allowed to equilibrate to room temperature prior to opening the pouch to minimize condensation.

4. I did not use all the strip wells supplied with my ELISA-ONE kit. How should I store the remaining strip wells?

Unused strip wells should be removed from the frame, placed back in their foil pouch, sealed with tape, and stored at +4°C or room temperature. Unused strip wells should be used within one month from the date of opening.

5. Does the ELISA-ONE microplate need to be blocked before use?

No, the ELISA-ONE microplate does not need to be blocked. ELISA-ONE plates are pre-coated and ready to use. However, for optimal performance, rinse the wells with sterile H2O to remove preservatives prior to use.

6. Which cell types are compatible with ELISA-ONE?

ELISA-ONE can be used for many adherent and nonadherent cell types, including transfected cell lines and primary cells. However, because kinase expression and phosphorylation conditions can vary from one cell line to another, some cells may be more amenable than others. Parameters such as stimulation time and cell number should be optimized for each cell line used. When using overexpressed intracellular targets, ensure full‐length expression of the target for correct binding of the antibody pair. The concentration of cell lysate should be optimized to ensure that the signal is within the working range of the assay.

7. Is cell culture supernatant compatible with ELISA-ONE?

ELISA-ONE works with many matrices, although sensitivity may be affected. Compatibility should be assessed on a case by case basis.

8. What is the 5X Lysis Buffer composition?

The 5X Lysis Buffer provided for the ELISA-ONE is a 5X stock that contains a combination of detergents, phosphatase inhibitors, salts and buffers. The 5X Lysis Buffer can be supplemented with Enhancer Solution to yield a versatile lysis solution that can be applied to many cells and tissues.

Lysing cells can yield to viscous lysate solutions difficult to handle, especially when concentrated lysates are required. Care should be taken when transferring lysates in order to minimize pipetting related variability.

9. Can I add extra components to the Lysis Mix?

Supplementation of the Lysis Mix with additional components (e.g. protease inhibitors, chelating agents, detergents) should be tested on a case by case basis for compatibility with ELISA‐ONE assays.

10. Are other lysis buffers (e.g. RIPA buffer) compatible with ELISA-ONE?

Optimal performance is ensured with the 5X Lysis Buffer provided with ELISA-ONE. Compatibility with other cell lysis buffers should be assessed on a case by case basis as these buffers could denature the ELISA-ONE antibody pair and affect the phosphorylation state of the target as well as its epitopes. Furthermore, strong detergents such as SDS are not recommended for use with ELISA‐ONE as they can also denature proteins.

11. Do I need a centrifugation step to lyse my cells?

Centrifugation is not required as the Lysis Mix effectively solubilizes the cells.

12. I have a limited amount / volume of sample. What is the lowest volume of lysate I can add on the ELISA-ONE plate?

ELISA‐ONE is optimized for 50μL of both cell lysate and Antibody Mix. However, if lysate availability is limited, as little as 25μL of lysate can be used, in combination with 50μL of Antibody Mix for a total volume of 75μL.

13. How do I measure the concentration of protein in the lysate?

We recommend using a BCA assay to measure the concentration of protein in the lysate, as this is compatible with the ELISA-ONE Extraction Buffer.

14. What does the Control Lysate consist of?

Control Lysates are lyophilized cell lysates, prepared from various cell types and stimulated to express the protein of interest. The Control Lysates are intended for use as a positive control for the assay only and should not be used for the absolute protein quantification.

15. The solution turned cloudy after the addition of the Antibody Mix. Does this affect the assay performance?

No, it does not. Cloudiness has sometimes been observed but has no effect on assay performance.

16. Are the antibody pairs sold separately?

No, the antibody pairs are sold exclusively within the ELISA-ONE kits.

17. The absorbance value of my ‘neat’ / undiluted sample is lower compared with absorbance values for the serial dilutions. Can you please explain what is happening?

You are experiencing a phenomenon known as the ‘hook effect’, which is an interference that affects many immunoassays. The hook effect happens when the concentration of the analyte is too high. This results in saturation of the capture and detection antibodies, and thus prevents the formation of the immunocomplex. The hook effect is often observed with tissue lysates. If experiencing the hook effect, it is recommended to perform serial dilutions of your sample until you find the linear range.

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