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PathScan® Total IKKα Sandwich ELISA Kit #7078
Figure 2. The relationship between the protein concentration of lysates from untreated and LPS-treated THP-1 cells and the absorbance at 450 nm using PathScan® Total IKKα Sandwich ELISA Kit is shown.Learn more about how we get our images
Figure 1. Treatment of differentiated THP-1 cells with LPS stimulates phosphorylation of IKKα at Ser176/180 as detected by the PathScan® Phospho-IKKα (Ser176/180) Sandwich ELISA Kit #7073 but does not affect the levels of total IKKα detected by PathScan® Total IKKα Sandwich ELISA Kit #7078. Differentiated THP-1 cells were treated with 1 μg/ml LPS for 10 minutes. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using IKKα Antibody #2682 (left) or Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb #2697 (right) are shown in the bottom figure.Learn more about how we get our images
Gallery: PathScan® Total IKKα Sandwich ELISA Kit #7078
- Figure 2. The relationship between the protein concentration of lysates from untreated and LPS-treated THP-1 cells and the absorbance at 450 nm using PathScan® Total IKKα Sandwich ELISA Kit is shown.
- Figure 1. Treatment of differentiated THP-1 cells with LPS stimulates phosphorylation of IKKα at Ser176/180 as detected by the PathScan® Phospho-IKKα (Ser176/180) Sandwich ELISA Kit #7073 but does not affect the levels of total IKKα detected by PathScan® Total IKKα Sandwich ELISA Kit #7078. Differentiated THP-1 cells were treated with 1 μg/ml LPS for 10 minutes. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using IKKα Antibody #2682 (left) or Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb #2697 (right) are shown in the bottom figure.
|Product Includes||Volume||Solution Color|
|IKKalpha Ab Coated Microwells||96 assays|
|IKKα Mouse Detection mAb||1 Ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 Ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml||Colorless|
|STOP Solution 7002||11 ml||Colorless|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 sheets|
|PathScan® Sandwich ELISA Lysis Buffer (1X) 7018||30 ml||Colorless|
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.
- 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L PBS: add 50 ml 10X PBS to 950 ml dH2O, mix.
- Bring all microwell strips to room temperature before use.
- Prepare 1X Wash Buffer by diluting 20X Wash Buffer (included in each PathScan® Sandwich ELISA Kit) in dH2O.
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.
- TMB Substrate: (#7004).
- STOP Solution: (#7002).
B. Preparing Cell Lysates
For adherent cells
- Aspirate media when the culture reaches 80–90% confluence. Treat cells by adding fresh media containing regulator for desired time.
- Remove media and rinse cells once with ice-cold 1X PBS.
- 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.
- Scrape cells off the plate and transfer to an appropriate tube. Keep on ice.
- Sonicate lysates on ice.
- 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
- 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.
- Collect cells by low speed centrifugation (~1,200 rpm) and wash once with 5–10 ml ice-cold 1X PBS.
- Cells harvested from 50 ml of growth media can be lysed in 2.0 ml of 1X cell lysis buffer plus 1 mM PMSF.
- Sonicate lysates on ice.
C. Test Procedure
- 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.
- 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.
- 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.
- Gently remove the tape and wash wells:
- Discard plate contents into a receptacle.
- Wash 4 times with 1X 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.
- Clean the underside of all wells with a lint-free tissue.
- Add 100 µl of detection antibody (green color) to each well. Seal with tape and incubate the plate at 37°C for 1 hr.
- Repeat wash procedure (Section C, Step 4).
- 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.
- Repeat wash procedure (Section C, Step 4).
- 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.
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.
- Read results
- Visual Determination: Read within 30 min after adding STOP solution.
- 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
The PathScan® Total IKKα Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of IKKα. An IKKα rabbit antibody has been coated onto the microwells. After incubation with cell lysates, IKKα (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, an IKKα mouse detection mAb is added to the captured phospho and nonphospho IKKα 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 this developed color is proportional to the quantity of total IKKα.
Antibodies in kit are custom formulations specific to kit.
The PathScan® IKKα Sandwich ELISA Kit from Cell Signaling Technology detects endogenous levels of total IKKα protein, 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).
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.