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|Application / Reactivity||Human||Mouse (Murine)|
|ELISA||14 ELISA Kits||14 ELISA Kits|
|Antigen||Signal Transducer and Activator of Transcription 4 (STAT4) ELISA Kits|
|Reactivity||Human, Mouse (Murine) Alternatives|
Kits with alternative reactivity to:
|Methode Type||Cell ELISA|
|3 references available|
|Supplier||Log in to see|
Product Details STAT4 ELISA KitTarget details Application Details Handling References for STAT4 Kit (ABIN1981841) Images
|Purpose||Semi-Quantitative Cell-Based Human/Mouse Stat 4 (Tyr693) Phosphorylation ELISA Kit for adherent whole cell lines.|
|Sample Type||Adherent Cell Culture|
|Specificity||The antibody pair provided in this kit recognizes human and mouse Stat 4 (pTyr693).|
96 wells (12 strips x 8 wells) Coated With Antibody
Wash Buffer Concentrate (20x)
Secondary Antibody or HRP Streptavidin
TMB One-Step Substrate Reagent
Cell Lysis Buffer
|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
Target detailsProduct Details STAT4 ELISA Kit Application Details Handling References for STAT4 Kit (ABIN1981841) Images back to top
|Alternative Name||STAT4 (STAT4 ELISA Kit Abstract)|
|Research Area||Chromatin and Nuclear Signaling, Transcription Factors, Signaling, Receptors, Inflammation|
Application DetailsProduct Details STAT4 ELISA Kit Target details Handling References for STAT4 Kit (ABIN1981841) Images back to top
Site and signal pathway-specificIn vitro detection of adherent cell cultureNo sample lysis neededCompatible with a standard ELISA plate readerFaster results than with ELISAAdaptable for high-throughput screening and drug discovery
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, Cat#: P4832) 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 manufac turer'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 anttibody (ITEM I-1 or I-2) into each well and incubate for 1 hour at room temperature.
NOTE: Item I-1 is the secondary antibody for Item G (primary antibody). Item I-2 is the secondary antibody for Item H (primary antibody).
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 STAT4 ELISA Kit Target details Application Details References for STAT4 Kit (ABIN1981841) 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 STAT4 Kit (ABIN1981841)Product Details STAT4 ELISA Kit Target details Application Details Handling Images back to top
|Product cited in:||
Kaplan, Sun, Hoey et al.: "Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice." in: Nature, Vol. 382, Issue 6587, pp. 174-7, 1996
Kaplan, Sun, Hoey et al.: "Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice." in: Nature, Vol. 382, Issue 6587, pp. 174-7, 1996 (PubMed).
Zhong, Wen, Darnell: "Stat3 and Stat4: members of the family of signal transducers and activators of transcription." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 91, Issue 11, pp. 4806-10, 1994 (PubMed).
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