AXL Protein, Mouse (HEK293, His-Fc)

The AXL protein has predicted roles in various cellular functions, binds to phosphatidylinositol 3-kinase and phosphatidylserine, and serves as a viral receptor. It negatively regulates apoptosis and positively regulates protein kinase B signaling. AXL Protein, Mouse (HEK293, His-Fc) is the recombinant mouse-derived AXL protein, expressed by HEK293 , with C-hFc, C-His labeled tag.

AXL protein is predicted to play a crucial role in various cellular functions, including phosphatidylinositol 3-kinase binding activity, phosphatidylserine binding activity, and virus receptor activity. Involved in negative regulation of apoptotic processes and positive regulation of protein kinase B signaling, AXL acts upstream of diverse processes such as animal organ development, myeloid cell homeostasis, and negative regulation of tumor necrosis factor production. Predicted to be located in cellular components like the actin cytoskeleton, cell surface, and host cell surface, AXL is expected to be an integral component of the plasma membrane and part of a receptor complex. With broad expression observed in various structures such as the alimentary system, brain, genitourinary system, respiratory system, and visual system, AXL likely plays a crucial role in numerous physiological processes across different tissues.

Immobilized AXL at 2 μg/mL (100 μL/well) can bind Biotinylated GAS6 protein. The ED₅₀ for this effect is 9.284 ng/mL, corresponding to a specific activity is 1.08×10^5 Unit/mg.

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  Immobilized AXL at 2 μg/mL (100 μL/well) can bind Biotinylated GAS6 protein. The ED₅₀ for this effect is 9.284 ng/mL, corresponding to a specific activity is 1.08×10⁵Unit/mg.

Materials
Biotin
Pre-chilled sterile water
Wash buffer: 1X PBST
Blocking buffer: 1% BSA in 1X PBST
TMB substrate solution: Mix substrate solution A and B at a 1:1 ratio immediately before use
Stop solution: 2 M H₂SO₄
AXL Protein, Mouse (HEK293, His-Fc) (HY-P74406)
GAS6 Protein, Human (HEK293, C-His) (HY-P70780A)

Procedure
Biotinylation
1. Dissolution of Biotin Reagent: Remove Biotin from -10 to -30°C and immediately dissolve 1 tube of Biotin in 180 µL of cold purified water. Gently pipette to mix and obtain a 10 mM Biotin solution.
2. Calculate the volume of EZ-Link^TM Sulfo-NHS-LC-LC-Biotin, No-Weigh^TM Format to be added.
3. Incubation: Incubate at room temperature in the dark for 45 minutes.
4. Quantification of Biotinylated Human GAS6: Use the BCA assay to quantify the protein concentration.
ELISA Detection
1. Coating: Dilute Fibronectin Protein to 10 µg/mL in 1X PBST, add 100 µL/well, and incubate overnight at 2-8°C.
2. Washing: Wash the plate with wash buffer (260 µL/well) , once, and pat dry.
3. Blocking: Add 100 µL/well of blocking buffer, incubate at 25°C with shaking at 450 rpm for 4 hours.
4. Dilution of Biotinylated Human GAS6: Prepare dilutions at concentrations of 50, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625, 0.1953125, 0.097656, 0.0488, 0.0244, 0.0122, 0 µg/mL.
5. Add Biotinylated Human GAS6: Add 100 µL/well, incubate at 25°C with shaking at 450 rpm for 2 hours.
6. Washing: Wash the plate with wash buffer (260 µL/well) , five times, with each wash lasting 1 minute, then pat dry.
7. Add secondary antibody: Add 100 µL/well, incubate at 25°C with shaking at 450 rpm for 1 hour.
8. Washing: Wash the plate with wash buffer (260 µL/well) , three times, with each wash lasting 1 minute, then pat dry.
9. Add TMB Substrate Solution: Add 100 µL/well, incubate at room temperature in the dark for 15 minutes.
10. Stop Reaction: Add 100 µL of 2 M H₂SO₄ per well.
11. Read Plate: Measure absorbance at 450 nm.
12. Using GraphPad Prism software, plot a curve with the OD value as the vertical axis and the Biotinylated Human GAS6 protein concentration as the horizontal axis, and calculate the ED₅₀ value.

Mouse

HEK293

C-hFc;C-His

NP_033491.2 (H20-P443)

26362  [NCBI]

Partial

HKDTQTEAGSPFVGNPGNITGARGLTGTLRCELQVQGEPPEVVWLRDGQILELADNTQTQVPLGEDWQDEWKVVSQLRISALQLSDAGEYQCMVHLEGRTFVSQPGFVGLEGLPYFLEEPEDKAVPANTPFNLSCQAQGPPEPVTLLWLQDAVPLAPVTGHSSQHSLQTPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQRPHHLHVVSRQPTELEVAWTPGLSGIYPLTHCNLQAVLSDDGVGIWLGKSDPPEDPLTLQVSVPPHQLRLEKLLPHTPYHIRISCSSSQGPSPWTHWLPVETTEGVPLGPPENVSAMRNGSQVLVRWQEPRVPLQGTLLGYRLAYRGQDTPEVLMDIGLTREVTLELRGDRPVANLTVSVTAYTSAGDGPWSLPVPLEPWRPGQGQPLHHLVSEPPPRAFSWP

95-120 kDa

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  Greater than 90% as determined by reducing SDS-PAGE.

Lyophilized powder.

Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4.

<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.