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REACTIVITY
H
Product Includes Volume Solution Color
Phospho-IKKbeta(S177/181) Ab Coated Microwells 96 tests
IKKbeta Detection Ab 11 ml Green
Anti-Mouse IgG HRP-Linked Antibody 11 ml Red
TMB Substrate 7004 11 ml Colorless
STOP Solution 7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
PathScan® Sandwich ELISA Lysis Buffer (1X) 7018 30 ml

Order Details

Custom Ordering Details: When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.
Page

ELISA Colormetric

NOTE: Refer to product-specific datasheets or product webpage for assay incubation temperature.

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 PBS: add 50 ml 10X PBS to 950 ml dH2O, mix.
  2. Bring all microwell strips to room temperature before use.
  3. Prepare 1X Wash Buffer by diluting 20X Wash Buffer (included in each PathScan® Sandwich ELISA Kit) in dH2O.
  4. 1X Cell Lysis Buffer: PathScan® Sandwich ELISA Lysis Buffer (#7018) 1X: This buffer is ready to use as is. 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.

    NOTE: Refer to product-specific datasheet or webpage for lysis buffer recommendation.

  5. TMB Substrate: (#7004).
  6. STOP Solution: (#7002).

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. Test Procedure

  1. After the microwell strips have reached room temperature, break off the required number of microwells. Place the microwells in the strip holder. Unused microwells must be resealed in the storage bag and stored at 4°C immediately.
  2. Cell lysates can be undiluted or diluted with sample diluent (supplied in each PathScan® Sandwich ELISA Kit, blue color). Individual datasheets or product webpage for each kit provide information regarding an appropriate dilution factor for lysates and kit assay results.
  3. Add 100 µl of each undiluted or diluted cell lysate to the appropriate well. Seal with tape and press firmly onto top of microwells. Incubate the plate for 2 hr at 37°C. Alternatively, the plate can be incubated overnight at 4°C.
  4. Gently remove the tape and wash wells:
    1. Discard plate contents into a receptacle.
    2. Wash 4 times with 1X wash buffer, 200 µl each time per well.
    3. 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.
    4. Clean the underside of all wells with a lint-free tissue.
  5. Add 100 µl of detection antibody (green color) to each well. Seal with tape and incubate the plate at 37°C for 1 hr.
  6. Repeat wash procedure (Section C, Step 4).
  7. Add 100 µl of HRP-linked secondary antibody (red color) to each well. Seal with tape and incubate the plate for 30 min at 37°C.
  8. Repeat wash procedure (Section C, Step 4).
  9. Add 100 µl of TMB substrate to each well. Seal with tape and incubate the plate for 10 min at 37°C or 30 min at 25°C.
  10. Add 100 µl of STOP solution to each well. Shake gently for a few seconds.

    NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP solution.

  11. Read results
    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 June 2005

revised November 2013

protocol id: 208

Product Description

The PathScan® Phospho-IKKβ (Ser177/181) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of IKKβ when phosphorylated at Ser177/181. A phospho-IKKβ (Ser177/181) rabbit mAb has been coated onto the microwells. After incubation with cell lysates, phospho-IKKβ (Ser177/181) protein is captured by the coated antibody. Following extensive washing, an IKKβ mouse detection mAb is added to detect the captured IKKβ protein. Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for the developed color is proportional to the quantity of IKKβ phosphorylated at Ser177/181.

Antibodies in kit are custom formulations specific to kit.


Specificity / Sensitivity

The PathScan® Phospho-IKKβ (Ser177/181) Sandwich ELISA Kit detects endogenous levels of IKKβ protein when phosphorylated at Ser177/181, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.


Species Reactivity: Human

The NF-κB/Rel transcription factors are present in the cytosol in an inactive state, complexed with the inhibitory IκB proteins (1-3). Most agents that activate NF-κB do so through a common pathway based on phosphorylation-induced, proteasome-mediated degradation of IκB (3-7). The key regulatory step in this pathway involves activation of a high molecular weight IκB kinase (IKK) complex whose catalysis is generally carried out by three tightly associated IKK subunits. IKKα and IKKβ serve as the catalytic subunits of the kinase and IKKγ serves as the regulatory subunit (8,9). Activation of IKK depends upon phosphorylation at Ser177 and Ser181 in the activation loop of IKKβ (Ser176 and Ser180 in IKKα), which causes conformational changes, resulting in kinase activation (10-13).


1.  Baeuerle, P.A. and Baltimore, D. (1988) Science 242, 540-6.

2.  Beg, A.A. and Baldwin, A.S. (1993) Genes Dev 7, 2064-70.

3.  Finco, T.S. et al. (1994) Proc Natl Acad Sci USA 91, 11884-8.

4.  Brown, K. et al. (1995) Science 267, 1485-8.

5.  Brockman, J.A. et al. (1995) Mol Cell Biol 15, 2809-18.

6.  Traenckner, E.B. et al. (1995) EMBO J 14, 2876-83.

7.  Chen, Z.J. et al. (1996) Cell 84, 853-62.

8.  Zandi, E. et al. (1997) Cell 91, 243-52.

9.  Karin, M. (1999) Oncogene 18, 6867-74.

10.  DiDonato, J.A. et al. (1997) Nature 388, 548-54.

11.  Mercurio, F. et al. (1997) Science 278, 860-6.

12.  Johnson, L.N. et al. (1996) Cell 85, 149-58.

13.  Delhase, M. et al. (1999) Science 284, 309-13.


Entrez-Gene Id 3551
Swiss-Prot Acc. O14920


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
PathScan® is a trademark of Cell Signaling Technology, Inc.