ST2/IL1RL1 Protein, Mouse (HEK293, His)

The ST2/IL1RL1 protein serves as a receptor for IL-33 and plays a crucial role in immunity and response to environmental stress through IL1RAP.It recruits MYD88, IRAK1/4 and TRAF6 upon stimulation, leading to MAPK phosphorylation.ST2/IL1RL1 Protein, Mouse (HEK293, His) is the recombinant mouse-derived ST2/IL1RL1 protein, expressed by HEK293 , with C-6*His labeled tag.

ST2/IL1RL1 serves as a receptor for interleukin-33 (IL-33), playing crucial roles in innate and adaptive immunity to maintain tissue homeostasis and respond to environmental stresses, in conjunction with its coreceptor IL1RAP. Upon stimulation, it recruits MYD88, IRAK1, IRAK4, and TRAF6, leading to the phosphorylation of various MAPKs, including MAPK3/ERK1, MAPK1/ERK2, MAPK14, and MAPK8. This receptor is implicated in helper T-cell function and, in the context of tissue injury, induces a UCP2-dependent mitochondrial rewiring. This rewiring attenuates the generation of reactive oxygen species, preserving the integrity of the Krebs cycle. This preservation is essential for sustained production of itaconate and subsequent GATA3-dependent differentiation of inflammation-resolving alternatively activated macrophages. Additionally, ST2/IL1RL1 plays a role in inhibiting IL-33 signaling.

1.Measured in a cell proliferation assay using A549 cells. The ED₅₀ for this effect is 34.04 ng/mL in the presence of 20 ng/mL recombinant mouse IL-33, corresponding to a specific activity is 2.51×10⁵ units/mg.
2.Immobilized Mouse ST2/IL1RL1 Protein at 2 μg/mL (100 μL/well) can bind Biotinylated Mouse IL-33, The ED₅₀ for this effect is 19.31 nM.

• 
  Measured in a cell proliferation assay using A549 cells. The ED₅₀ for this effect is 34.04 ng/mL in the presence of 20 ng/mL recombinant mouse IL-33, corresponding to a specific activity is 2.51×10⁵ units/mg.

Materials
Test protein: ST2/IL1RL1 Protein, Mouse (HEK293, His) (HY-P72459)
Ligand protein: IL-33 Protein, Mouse (HY-P7218)
Biotin
Pre-chilled Sterile Water
Washing buffer: 1×PBST
Blocking buffer: PBST with 1% BSA
TMB Chromogenic Solution: Mix Chromogenic Solution A and Chromogenic Solution B at a ratio of 1:1 before use, and prepare freshly as needed
Stop solution: 2 M H₂SO₄
HRP-Streptavidin

Procedure
Biotin Labeling
1. Dissolution of Biotin reagent: Take out Biotin from -10~-30℃, immediately dissolve 1 tube of Biotin with 180 μL of cold pure water. Gently pipette to mix well to prepare 10 mM Biotin.
2. Calculate the volume of EZ-Link^TM Sulfo-NHS-LC-LC-Biotin, No-Weigh^TM Format to be added.
3. Incubate at room temperature in the dark for 45 min.
4. Quantification of labeled protein: Use BCA assay for protein concentration quantification; the concentration of the labeled Biotinylated SARS-CoV-2 S1 is 208 μg/mL.

ELISA Detection
1. Coating: Dilute Mouse ST2/IL1RL1 to 2 μg/mL with 1×PBS, add 100 μL/well, and incubate overnight at 2-8℃.
2. Plate washing: Wash the plate with washing buffer, 260 μL per well, wash once, and pat dry.
3. Blocking: Use 1×PBST blocking buffer containing 1% BSA, add 150 μL/well, and block at 25℃ with 450 rpm shaking for 4 h.
4. Dilute Biotinylated SARS-CoV-2 S1 to the following concentrations: 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000, 2000 ng/mL.
5. Add receptor protein: 100 μL/well, incubate at 25℃ with 450 rpm shaking for 2 h.
6. Plate washing: Wash the plate with washing buffer, 260 μL per well, wash 5 times with 1 min retention each time, then pat dry.
7. Add secondary antibody: Dilute the secondary antibody at a ratio of 1:2000, add 100 μL per well, and incubate at 25℃ with 450 rpm shaking for 1 h.
8. Plate washing: Wash the plate with washing buffer, 260 μL/well, wash 3 times with 1 min retention each time, and pat dry.
9. Add TMB chromogenic solution: 100 μL per well, incubate at room temperature in the dark for 15 min.
10. Terminate the reaction: Add 2 M H₂SO₄, 100 μL/well.
11. Plate reading: Read the absorbance at 450 nm.
12. Use GraphPad Prism software to plot a curve with OD value as the ordinate and Biotinylated SARS-CoV-2 S1 concentration as the abscissa, and calculate the ED₅₀ value.

Mouse

HEK293

C-6*His

P14719-2 (S27-A337)

17082  [NCBI]

Partial

SKSSWGLENEALIVRCPQRGRSTYPVEWYYSDTNESIPTQKRNRIFVSRDRLKFLPARVEDSGIYACVIRSPNLNKTGYLNVTIHKKPPSCNIPDYLMYSTVRGSDKNFKITCPTIDLYNWTAPVQWFKNCKALQEPRFRAHRSYLFIDNVTHDDEGDYTCQFTHAENGTNYIVTATRSFTVEEKGFSMFPVITNPPYNHTMEVEIGKPASIACSACFGKGSHFLADVLWQINKTVVGNFGEARIQEEEGRNESSSNDMDCLTSVLRITGVTEKDLSLEYDCLALNLHGMIRHTIRLRRKQPSKECPSHIA

Approximately 50-78 kDa, based on SDS-PAGE under reducing condition, due to the glycosylation.

• 
  ≥ 95%, as determined by reducing SDS-PAGE.

Lyophilized powder

1.Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4.
2.Lyophilized from a 0.22 μm filtered solution of 20 mM PB, 150 mM NaCl, pH 7.4.
Please refer to the lot-specific COA for specific buffer information.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.