JNK Kit ELISA

N° du produit ABIN1981833

Détail du produit JNK Kit ELISA

Antigène: JNK (MAPK8) (Mitogen-Activated Protein Kinase 8 (MAPK8))

Épitope: pThr183, pTyr185

Reactivité: Humain, Souris, Rat

Méthode de détection: Colorimetric

Type de méthode: Cell ELISA

Application: ELISA

Fonction: Cell-Based Human/Mouse/Rat JNK (Thr183/Tyr185) Phosphorylation ELISA Kit. Suitable for adherent whole cell lines.

Marque: CellBIND®

Type d'échantillon: Cell Culture Cells

Analytical Method: Semi-Quantitative

Specificité: The antibodies provided in this kit recognizes human, mouse and rat JNK phosphorylated at sites Thr183 and Tyr185 as well as total JNK for comparison.

Attributs du produit:

• Site and signal pathway-specific
• In vitro detection of adherent cell culture
• No sample lysis needed
• Compatible with a standard ELISA plate reader
• Faster results than with ELISA
• Adaptable for high-throughput screening and drug discovery

Ingrédients:

• uncoated 96-well Microplate
• Wash Buffer A
• Wash Buffer B
• Fixing Solution
• Quenching Buffer
• Blocking Buffer
• Anti-phospho antibody
• Anti-pan antibody
• HRP-Conjugated Secondary Antibody
• TMB One-Step Substrate
• Stop Solution

Matériel non inclus:

• Distilled or deionized water
• 100 mL and 1 liter graduated cylinders
• Tubes to prepare sample dilutions
• Protease and Phosphatase inhibitors
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Adjustable 1-25 mL pipettes for reagent preparation
• Benchtop rocker or shaker
• Microplate reader capable of measuring absorbance at 450 nm

Information d'application

Volume d'échantillon: 100 μL

Plaque: Uncoated

Protocole:

1. Seed 10,000-30,000 cells into each well and incubate overnight.
2. Apply various treatment, inhibitors or activators according to manufacture's instructions.
3. Add 100 μL of Fixing Solution into each well and incubate for 20 min at RT with shaking.
4. Add 200 μL of prepared 1X Quenching Buffer and incubate 20 min at RT.
5. Add 200 μL of Blocking Solution and incubate for 1 h at 37 °C.
6. Add 50 μL of 1X anti-phospho-protein specific antibody or anti-pan-protein specific antibody to each well and incubate for 2 h at RT.
7. Add 50 μL of prepared 1X HRP-Anti-Rabbit or Mouse IgG and incubate for 1 h at RT.
8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 μL of Stop Solution to each well.
11. Read at 450 nm immediately.

Préparation des réactifs:

NOTE: Thaw all reagents to room temperature immediately before use. If wash buffers contain visible crystals, warm to room temperature and mix gently until dissolved.
NOTE: Briefly centrifuge (~1,000g) ITEMS G, H, and I before opening to ensure maximum recovery.
For more information look at the picture.

Procédure de l'essai:

NOTE: ALL incubations and wash steps must be performed under gentle rocking or rotation (~1-2 cycles/sec).
1. Design your experiment. For example, see Figure 2 below.
OPTIONAL: If seeding HUVECs, HMEC-1 or other loosely attached cells, coat the Uncoated 96-Well Microplate (ITEM A) by adding 100 µL poly-L-Lysine (Recommended Sigma Aldrich) into each well and then follow manufacturer's instructions. A pre-coated CellBIND® microplate or other poly-lysine treated tissue culture plate may be used in place of Item A.
2. Seed 100 µL of 30,000 cells into each well of the Uncoated 96-Well Microplate (ITEM A) provided and incubate overnight at 37 °C with 5 % CO2.
NOTE: The optimal cell number used will vary on the cell line and the relative amount of protein phosphorylation. More or less cells may be used but this must be determined empirically.
NOTE: The cells can be starved ~4-24 hours (depending on cell line) prior to treatment with inhibitors or activators.
3. Apply various treatments, inhibitors (such as siRNA or chemicals) or activators according to manufacturer's instructions and incubate for the desired time points.
NOTE: It is recommended to dissolve inhibitors or activators into serum-free cell culture medium before treating the cells (unless otherwise stated in the manufacturer's instructions.)
4. Discard the cell culture medium by flipping the microplate upside down and gently tapping the bottom of the microplate over a sink.
5. Wash by pipetting 200 µL of the prepared 1X Wash Buffer A (ITEM B) into each well. Discard the wash buffer (same as step 4) and wash 2 more times for a total of 3 washes using fresh wash buffer each time. After the final wash, gently blot the microplate onto a paper towel to remove any excess/remaining buffer.
NOTE: To avoid cell loss, do not pipette directly onto the cells. Instead, gently dispense the liquid down the wall of cell culture wells. Also avoid the use of vacuum suction or too forcefully tapping the microplate when discarding any solution.
6. Add 100 µL of Fixing Solution (ITEM D) into each well and incubate for 20 minutes at room temperature.
NOTE: The fixing solution is used to permeabilize the cells.
7. Repeat wash step 5.
8. Add 200 µL of the prepared 1X Quenching Buffer (ITEM E) into each well and incubate 20 minutes at room temperature.
NOTE: The quenching buffer is used to minimize the background response.
9. Wash 4 times with 1X Wash Buffer A.
10. Add 200 µL of the prepared 1X Blocking Buffer (ITEM F) into each well and incubate for 1 hour at 37 °C.
11. Wash 3 times with the prepared 1X Wash Buffer B (ITEM C).
NOTE: If needed, the microplate may be stored at -80 °C for several days after this wash.
12. Add 50 µL of the prepared 1X primary antibody (ITEM G or H) into each corresponding well and incubate for 2 hours at room temperature.
13. Wash 4 times with 1X Wash Buffer B.
14. Add 50 µL of the prepared 1X HRP Conjugated secondary antibody (ITEM I) into each well and incubate for 1 hour at room temperature.
15. Wash 4 times with 1X Wash Buffer B.
16. Add 100 µL of the TMB Substrate (ITEM J) into each well and incubate for 30 minutes at room temperature in the dark.
17. Add 50 µL of the Stop Solution (ITEM K) into each well. Read at 450 nm immediately.

Restrictions: For Research Use only

Stockage

Conseil sur la manipulation: Avoid repeated freeze-thaw cycles.

Stock: -20 °C

Stockage commentaire: The entire kit may be stored at -20°C for up to 6 months from the date of shipment. Avoid repeated freeze-thaw cycles.

Date de péremption: 6 months

Références pour JNK Kit ELISA (ABIN1981833)

Su, Zhou, Tao, Guo, Guo, Zhang, Zhang, Huang, Tang, Dong, Hu: "G-CSF protects human brain vascular endothelial cells injury induced by high glucose, free fatty acids and hypoxia through MAPK and Akt signaling." dans: PLoS ONE, Vol. 10, Issue 4, pp. e0120707, (2015) (PubMed).

Fleming, Armstrong, Morrice, Paterson, Goedert, Cohen: "Synergistic activation of stress-activated protein kinase 1/c-Jun N-terminal kinase (SAPK1/JNK) isoforms by mitogen-activated protein kinase kinase 4 (MKK4) and MKK7." dans: The Biochemical journal, Vol. 352 Pt 1, Issue 3, pp. 145-54, (2001) (PubMed).

Hinton, Henderson, Blanks, Rudnicka, Miller: "Monoclonal antibodies react with neuronal subpopulations in the human nervous system." dans: The Journal of comparative neurology, Vol. 267, Issue 3, pp. 398-408, (1988) (PubMed).

De Gregori, Magrassi: "[Biliary calculosis in gastric resections for ulcer]." dans: Il Fegato, Vol. 11, Issue 2, pp. 201-9 (PubMed).

Détail du antigène

Antigène: JNK (MAPK8) (Mitogen-Activated Protein Kinase 8 (MAPK8))

Autre désignation: JNK (MAPK8 Produits)

Synonymes: JNK Kit ELISA, JNK-46 Kit ELISA, JNK1 Kit ELISA, JNK1A2 Kit ELISA, JNK21B1/2 Kit ELISA, PRKM8 Kit ELISA, SAPK1 Kit ELISA, SAPK1c Kit ELISA, AI849689 Kit ELISA, Prkm8 Kit ELISA, BSK Kit ELISA, BSK/DJNK Kit ELISA, Bsk Kit ELISA, CG5680 Kit ELISA, D-JNK Kit ELISA, D-junk Kit ELISA, DBSK/JNK Kit ELISA, DJNK Kit ELISA, DJNK/bsk Kit ELISA, Dmel\\CG5680 Kit ELISA, JNK/SAPK Kit ELISA, Jnk Kit ELISA, Junk Kit ELISA, SAPKa Kit ELISA, c-Jun Kit ELISA, dJNK Kit ELISA, jnk Kit ELISA, T10F20.15 Kit ELISA, jnk1 Kit ELISA, sapk1 Kit ELISA, mapk8 Kit ELISA, zgc:112379 Kit ELISA, JNKb Kit ELISA, mapk8b Kit ELISA, mitogen-activated protein kinase 8 Kit ELISA, Stress-activated protein kinase jnk-1 Kit ELISA, basket Kit ELISA, Protein kinase superfamily protein Kit ELISA, mitogen-activated protein kinase 8 L homeolog Kit ELISA, mitogen-activated protein kinase 8b Kit ELISA, mitogen-activated protein kinase 8B-like Kit ELISA, MAPK8 Kit ELISA, jnk-1 Kit ELISA, Mapk8 Kit ELISA, bsk Kit ELISA, ATMPK8 Kit ELISA, mapk8.L Kit ELISA, mapk8b Kit ELISA, LOC109094263 Kit ELISA

ID gène: 5599

UniProt: P45983

Pathways: Signalisation MAPK, Signalisation WNT, Signalisation TLR, Fc-epsilon Receptor Signaling Pathway, Neurotrophin Signaling Pathway, Activation of Innate immune Response, Hepatitis C, Toll-Like Receptors Cascades, Signaling of Hepatocyte Growth Factor Receptor, S100 Proteins