We are preparing the requested document. Please wait, this may take a while...!
|Antigen||Mitogen-Activated Protein Kinase 8 (MAPK8) ELISA Kits|
|Reactivity||Human, Mouse (Murine), Rat (Rattus) Alternatives|
Kits with alternative reactivity to:
|Methode Type||Cell ELISA|
|4 references available|
|Supplier||Log in to see|
Product Details JNK ELISA KitTarget details Application Details Handling References for JNK Kit (ABIN1981833) Images
|Purpose||Semi-Quantitative Cell-Based Human/Mouse/Rat JNK (Thr183/Tyr185) Phosphorylation ELISA Kit for adherent whole cell lines.|
|Sample Type||Adherent Cell Culture|
Protein phosphorylation is instrumental in the regulation of protein activity within a cell. It plays important roles in the living cells including proliferation, differentiation and metabolism. A large number of protein kinases and phosphatases have been extensively investigated, and have been shown to be involved in signal transduction pathways.
The Cell-Based Human/Mouse/Rat Cell-Based JNK (Thr183/Tyr185) ELISA kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in cells. It can be used for measuring the relative amount of JNK (Thr183/Tyr185) phosphorylation and screening the effects of various treatments, inhibitors (such as siRNA or chemicals), or activators in cultured human, mouse and rat cell lines. By determining JNK protein phosphorylation in your experimental model system, you can verify pathway activation in your cell lines without spending excess time and effort in preparing cell lysate and performing an analysis of Western Blot. In the Cell-Based JNK (Thr183/Tyr185) ELISA kit, cells are seeded into a 96 well tissue culture plate. The cells are fixed after various treatments, inhibitors or activators. After blocking, Anti- Phospho-JNK (Thr183/Tyr185) or Anti-JNK is pipetted into the wells and incubated. The wells are washed, and HRP-conjugated anti-mouse IgG is added to the wells. The wells are washed again, a TMB substrate solution is added to the wells and color develops in proportion to the amount of protein. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
A, Uncoated 96-Well Microplate, 1 plate, Room Temperature
B, 20X Wash Buffer A Concentrate, 1 vial (30 ml), 2-8 °C
C, 20X Wash Buffer B Concentrate, 1 vial (30 ml), 2-8 °C
D, Fixing Solution, 1 vial (30 ml), 2-8 °C
E, 30X Quenching Buffer Concentrate, 1 vial (2 ml), 2-8 °C
F, 5X Blocking Buffer Concentrate, 1 vial (20 ml) 2-8 °C (1 month)
G, 1000X Mouse Anti-phospho (Thr183/Tyr185) JNK Concentrate, 1 vial (7 μl), -20 °C
H, 1000X Mouse Anti-JNK Concentrate, 1 vial (7 µL ), -20 °C
I, 1000X HRP Conjugated Anti-Mouse IgG Concentrate, 1 vial (10 µL ), -20 °C
J, TMB Substrate, 1 vial (12 ml), 2-8 °C
K, Stop Solution**, 1 vial (14 ml), 2-8 °C
*For up to 3 months (unless otherwise stated) or until expiration date.
**Contains 0.2 M Sulfuric Acid
|Material not included||
1. A model cell line, protein tyrosine kinase inhibitors, growth factors or cytokines
2. Microplate reader capable of measuring absorbance at 450 nm
3. 37 °C incubator
4. Precision pipettes to deliver 2 µL to 1 ml volumes
5. Adjustable 1-25 ml pipettes for reagent preparation
6. 100 ml and 1 liter graduated cylinders
7. Absorbent paper
8. Distilled or deionized water
9. Orbital shaker or oscillating rocker
Target detailsProduct Details JNK ELISA Kit Application Details Handling References for JNK Kit (ABIN1981833) Images back to top
|Alternative Name||JNK (MAPK8 ELISA Kit Abstract)|
|Pathways||MAPK Signaling, WNT Signaling, TCR Signaling, TLR Signaling, Fc-epsilon Receptor Signaling Pathway, Neurotrophin Signaling Pathway|
Application DetailsProduct Details JNK ELISA Kit Target details Handling References for JNK Kit (ABIN1981833) Images back to top
Quality control: Representative results of Cell-Based JNK (Thr183/Tyr185) are shown below:
1. Add cells
2. Treatment with stimulators or inhibitors
3. Fixing and blocking
4. Anti-phospho-protein antibody or anti-pan-protein antibody
5. HRP-conjugated secondary antibody
6. Develop with substrate
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.
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|
HandlingProduct Details JNK ELISA Kit Target details Application Details References for JNK Kit (ABIN1981833) Images back to top
|Handling Advice||Avoid repeated freeze-thaw cycles.|
|Storage Comment||Store entire kit at ≤ -20 °C immediately upon arrival.|
|Expiry Date||6 months|
References for JNK Kit (ABIN1981833)Product Details JNK ELISA Kit Target details Application Details Handling Images back to top
|Product cited in:||
Su, Zhou, Tao et al.: "G-CSF protects human brain vascular endothelial cells injury induced by high glucose, free fatty acids and hypoxia through MAPK and Akt signaling." in: PLoS ONE, Vol. 10, Issue 4, pp. e0120707, 2015 (PubMed).
De Gregori, Magrassi: "[Biliary calculosis in gastric resections for ulcer]." in: Il Fegato, Vol. 11, Issue 2, pp. 201-9 (PubMed).
Hinton, Henderson, Blanks et al.: "Monoclonal antibodies react with neuronal subpopulations in the human nervous system." in: The Journal of comparative neurology, Vol. 267, Issue 3, pp. 398-408, 1988
Hinton, Henderson, Blanks et al.: "Monoclonal antibodies react with neuronal subpopulations in the human nervous system." in: The Journal of comparative neurology, Vol. 267, Issue 3, pp. 398-408, 1988 (PubMed).
ImagesProduct Details JNK ELISA Kit Target details Application Details Handling References for JNK Kit (ABIN1981833) back to top