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7836
PathScan® Total NF-κB p65 Sandwich ELISA Antibody Pair
ELISA Kits
ELISA Antibody Pair

PathScan® Total NF-κB p65 Sandwich ELISA Antibody Pair #7836

Citations (0)
The relationship between lysate protein concentration from untreated and TNF-α and IL-1β treated HeLa cells and the absorbance at 450 nm using PathScan® Total NF-κB p65 Sandwich ELISA Antibody Pair #7836 is shown. HeLa cells were treated with TNF-α and IL-1β for 5 minutes at 37ºC and then lysed.
To Purchase # 7836
Cat. # Size Qty. Price
7836S
1 Kit  (Reagents for 4 x 96 well plates)

Important Ordering Details

Custom Ordering Details: Product is assembled to order. Please allow up to three business days for your order to be processed.

Supporting Data

REACTIVITY H M

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected
Product Includes Volume Cap Color
NF-kB p65 Capture Rabbit mAb (100X) 400 µl Pink
NF-kB p65 Detection Mouse mAb (100X) 400 µl Blue
Anti-mouse IgG, HRP-linked Antibody (1000X) 40 µl Yellow

Storage

Capture and detection antibodies are stored at 4°C. HRP-linked secondary reagent is stored at -20°C.

Protocol

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ELISA Antibody Pair

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. Wash Buffer: 1X PBS/0.05% Tween® 20, (20X PBST #9809).
  3. Blocking Buffer: 1X PBS/0.05% Tween® 20, 1% BSA.
  4. 1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis Buffer to 9 ml of dH2O, mix. Buffer can be stored at 4°C for short-term use (1–2 weeks).

    Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) (#8553) immediately before use.

  5. Bovine Serum Albumin (BSA): (#9998).
  6. TMB Substrate: (#7004).
  7. STOP Solution: (#7002)

    NOTE: Reagents should be made fresh daily.

B. Preparing Cell Lysates

For adherent cells

  1. Aspirate media when the culture reaches 80–90% confluence. Treat cells by adding fresh media containing regulator for desired time.
  2. Remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 ml ice-cold 1X Cell Lysis Buffer plus 1 mM PMSF to each plate (10 cm diameter) and incubate the plate on ice for 5 min.
  4. Scrape cells off the plate and transfer to an appropriate tube. Keep on ice.
  5. Sonicate lysates on ice.
  6. Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at -80°C in single-use aliquots.

For suspension cells

  1. Remove media by low speed centrifugation (~1,200 rpm) when the culture reaches 0.5–1.0 x 106 viable cells/ml. Treat cells by adding fresh media containing regulator for desired time.
  2. Collect cells by low speed centrifugation (~1,200 rpm) and wash once with 5–10 ml ice-cold 1X PBS.
  3. Cells harvested from 50 ml of growth media can be lysed in 2.0 ml of 1X cell lysis buffer plus 1 mM PMSF.
  4. Sonicate lysates on ice.
  5. Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Store at -80°C in single-use aliquots.

C. Coating Procedure

  1. Rinse microplate with 200 µl of dH2O, discard liquid. Blot on paper towel to make sure wells are dry.
  2. Dilute capture antibody 1:100 in 1X PBS. For a single 96 well plate, add 100 µl of capture antibody stock to 9.9 ml 1X PBS. Mix well and add 100 µl/well. Cover plate and incubate overnight at 4°C (17–20 hr).
  3. After overnight coating, gently uncover plate and wash wells:
    1. Discard plate contents into a receptacle.
    2. Wash four times with wash buffer, 200 µl each time per well. For each wash, strike plates on fresh paper towels hard enough to remove the residual solution in each well, but do not allow wells to completely dry at any time.
    3. Clean the underside of all wells with a lint-free tissue.
  4. Block plates. Add 150 µl of blocking buffer/well, cover plate, and incubate at 37°C for 2 hr.
  5. After blocking, wash plate (Section C, Step 3). Plate is ready to use.

D. Test Procedure

  1. Lysates can be used undiluted or diluted in blocking buffer. 100 µl of lysate is added per well. Cover plate and incubate at 37°C for 2 hr.
  2. Wash plate (Section C, Step 3).
  3. Dilute detection antibody 1:100 in blocking buffer. For a single 96 well plate, add 100 µl of detection antibody Stock to 9.9 ml of blocking buffer. Mix well and add 100 µl/well. Cover plate and incubate at 37°C for 1 hr.
  4. Wash plate (Section C, Step 3).
  5. Secondary antibody, either streptavidin anti-mouse or anti-rabbit-HRP, is diluted 1:1000 in blocking buffer. For a single 96 well plate, add 10 µl of secondary antibody stock to 9.99 ml of blocking buffer. Mix well and add 100 µl/well. Cover and incubate at 37°C for 30 min.
  6. Wash plate (Section C, Step 3).
  7. Add 100 µl of TMB substrate per well. Cover and incubate at 37°C for 10 min.
  8. Add 100 µl of STOP solution per well. Shake gently for a few seconds.
  9. Read plate on a microplate reader at absorbance 450 nm.
    1. Visual Determination: Read within 30 min after adding STOP solution.
    2. Spectrophotometric Determination: Wipe underside of wells with a lint-free tissue. Read absorbance at 450 nm within 30 min after adding STOP solution.

posted January 2008

revised Sepetember 2013

Protocol Id: 20

Product Description

CST's PathScan® Total NF-κB p65 Sandwich ELISA Antibody Pair is offered as an economical alternative to our PathScan® Total NF-κB p65 Sandwich ELISA Kit #7174. Capture and Detection antibodies (100X stocks) and HRP-conjugated secondary antibody (1000X stock) are supplied. Sufficient reagents are supplied for 4 x 96 well ELISAs. The NF-κB p65 Capture Antibody is coated in PBS overnight in a 96 well microplate. After blocking, cell lysates are added followed by a NF-κB p65 Detection Antibody and anti-Mouse IgG, HRP conjugated antibody. HRP substrate, TMB, is added for color development. The magnitude of the absorbance for this developed color is proportional to the quantity of total NF-κB p65 protein.
Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

For Antibody Pair specificity and sensitivity, please refer to the corresponding PathScan® Sandwich ELISA Kit. Note: This antibody pair detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Species Reactivity:

Human, Mouse

Background

Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammatory and immune responses (1,2). There are five family members in mammals: RelA, c-Rel, RelB, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Both p105 and p100 are proteolytically processed by the proteasome to produce p50 and p52, respectively. Rel proteins bind p50 and p52 to form dimeric complexes that bind DNA and regulate transcription. In unstimulated cells, NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins (3-5). NF-κB-activating agents can induce the phosphorylation of IκB proteins, targeting them for rapid degradation through the ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus where it regulates gene expression (6-8). NIK and IKKα (IKK1) regulate the phosphorylation and processing of NF-κB2 (p100) to produce p52, which translocates to the nucleus (9-11).

Pathways

Explore pathways related to this product.

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not for Use in Diagnostic Procedures.
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U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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