MAPK14 ELISA Kit

Catalog No. ABIN1981835

Product Details MAPK14 ELISA Kit

Target: MAPK14 (Mitogen-Activated Protein Kinase 14 (MAPK14))

Binding Specificity: pThr180, pTyr182

Reactivity: Human, Rat, Mouse

Detection Method: Colorimetric

Method Type: Cell ELISA

Application: ELISA

Purpose: Cell-Based Human/Mouse/Rat MAPK (Thr180/Tyr182) Phosphorylation ELISA Kit. Suitable for adherent whole cell lines.

Brand: CellBIND®

Sample Type: Cell Culture Cells

Analytical Method: Semi-Quantitative

Specificity: The antibodies provided in this kit recognizes human, mouse and rat P38 MAPK phosphorylated at sites Thr180 and pTyr182 as well as total MAPK for comparison.

Characteristics:

• 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

Components:

• 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

Material not included:

• 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

Application Details

Sample Volume: 100 μL

Plate: Uncoated

Protocol:

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.

Reagent Preparation:

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.

Assay Procedure:

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

Handling

Handling Advice: Avoid repeated freeze-thaw cycles.

Storage: -20 °C

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

Expiry Date: 6 months

References for MAPK14 ELISA Kit (ABIN1981835)

Hale, Oyler, Swaminathan, Ahmed: "Basic tetrapeptides as potent intracellular inhibitors of type A botulinum neurotoxin protease activity." in: The Journal of biological chemistry, Vol. 286, Issue 3, pp. 1802-11, (2011) (PubMed).

Morinobu, Gadina, Strober, Visconti, Fornace, Montagna, Feldman, Nishikomori, OShea: "STAT4 serine phosphorylation is critical for IL-12-induced IFN-gamma production but not for cell proliferation." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 99, Issue 19, pp. 12281-6, (2002) (PubMed).

Winston, Chan, Johnson, Riches: "Activation of p38mapk, MKK3, and MKK4 by TNF-alpha in mouse bone marrow-derived macrophages." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 159, Issue 9, pp. 4491-7, (1997) (PubMed).

Han, Lee, Bibbs, Ulevitch: "A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells." in: Science (New York, N.Y.), Vol. 265, Issue 5173, pp. 808-11, (1994) (PubMed).

Target Details for MAPK14

Target: MAPK14 (Mitogen-Activated Protein Kinase 14 (MAPK14))

Alternative Name: p38 (MAPK14 Products)

Synonyms: CSBP ELISA Kit, CSBP1 ELISA Kit, CSBP2 ELISA Kit, CSPB1 ELISA Kit, EXIP ELISA Kit, Mxi2 ELISA Kit, PRKM14 ELISA Kit, PRKM15 ELISA Kit, RK ELISA Kit, SAPK2A ELISA Kit, p38 ELISA Kit, p38ALPHA ELISA Kit, CRK1 ELISA Kit, Csbp1 ELISA Kit, Csbp2 ELISA Kit, Exip ELISA Kit, Hog ELISA Kit, Prkm14 ELISA Kit, Prkm15 ELISA Kit, Sapk2A ELISA Kit, p38Hog ELISA Kit, p38alpha ELISA Kit, p38b ELISA Kit, zp38b ELISA Kit, MAPK14 ELISA Kit, 186F5S ELISA Kit, BG:DS00797.3 ELISA Kit, CG7393 ELISA Kit, D-p38 ELISA Kit, D-p38 MAPK ELISA Kit, D-p38b ELISA Kit, Dm p38b ELISA Kit, Dmel\\CG7393 ELISA Kit, Dmp38b ELISA Kit, Dp38 ELISA Kit, Dp38b ELISA Kit, ESTS:186F5S ELISA Kit, Mpk34C ELISA Kit, anon-sts23 ELISA Kit, dp38b ELISA Kit, p38 MAPK ELISA Kit, p38 beta ELISA Kit, p38B ELISA Kit, p38Kb ELISA Kit, p38beta ELISA Kit, Crk1 ELISA Kit, p38-alpha ELISA Kit, p38MAPK ELISA Kit, p38a ELISA Kit, csbp ELISA Kit, mapk14a ELISA Kit, mxi2 ELISA Kit, sapk2 ELISA Kit, sapk2a ELISA Kit, AP22.98 ELISA Kit, AP22_98 ELISA Kit, ATMPK14 ELISA Kit, mitogen-activated protein kinase 14 ELISA Kit, SAPK2a ELISA Kit, MAP kinase 14A ELISA Kit, MAP kinase p38a ELISA Kit, MAPK 14A ELISA Kit, fk28c03 ELISA Kit, hm:zeh1243 ELISA Kit, wu:fk28c03 ELISA Kit, zp38a ELISA Kit, P38C-CRK ELISA Kit, mitogen-activated protein kinase 14 ELISA Kit, mitogen activated protein kinase 14 ELISA Kit, mitogen-activated protein kinase 14b ELISA Kit, p38b MAP kinase ELISA Kit, mitogen-activated protein kinase 14 S homeolog ELISA Kit, mitogen-activated protein kinase 14a ELISA Kit, CRK proto-oncogene, adaptor protein ELISA Kit, MAPK14 ELISA Kit, Mapk14 ELISA Kit, mapk14b ELISA Kit, p38b ELISA Kit, mapk14.S ELISA Kit, MPK14 ELISA Kit, mapk14a ELISA Kit, CRK ELISA Kit

Background: P38

Gene ID: 1432

UniProt: Q16539

Pathways: MAPK Signaling, Neurotrophin Signaling Pathway, Activation of Innate immune Response, Cellular Response to Molecule of Bacterial Origin, Regulation of Muscle Cell Differentiation, Regulation of Cell Size, Hepatitis C, Toll-Like Receptors Cascades, Autophagy, Thromboxane A2 Receptor Signaling, BCR Signaling, S100 Proteins