CD157 Protein, Rat (HEK293, His)

CD157 protein catalyzes the synthesis of cADPR from NAD(+) and hydrolyzes cADPR to ADPR. cADPR acts as a second messenger, releasing calcium from intracellular stores. CD157 protein may also contribute to pre-B cell growth. CD157 Protein, Rat (HEK293, His) is the recombinant rat-derived CD157 protein, expressed by HEK293 , with C-His labeled tag.

CD157 protein acts as an enzyme that catalyzes the synthesis of cyclic ADP-beta-D-ribose (cADPR) from NAD(+) and also hydrolyzes cADPR to ADP-D-ribose (ADPR). Cyclic ADPR functions as a natural second messenger, triggering the release of calcium from intracellular stores and thereby controlling the mobilization of intracellular calcium. Additionally, CD157 protein may play a role in the growth of pre-B cells.

Measured by its binding ability in a functional ELISA. When Fibronectin is immobilized at 10 µg/mL (100 µL/well) can bind Biotinylated Rat CD157 Protein. The ED₅₀ for this effect is 4.274 μg/mL.

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  Measured by its binding ability in a functional ELISA. When Fibronectin is immobilized at 10 µg/mL (100 µL/well) can bind Biotinylated Rat CD157 Protein. The ED₅₀ for this effect is 4.274 μg/mL.

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
CD157 Protein, Rat (HEK293, His) (HY-P76205)
Fibronectin Protein

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. Calculation of EZ-Link^TM Sulfo-NHS-LC-LC-Biotin, No-Weigh^TM Format Volume: Calculate the volume to be added.
3. Incubation: Incubate at room temperature in the dark for 45 minutes.
4. Quantification of Biotinylated Rat CD157 Protein: Use the BCA assay to quantify the protein concentration. The concentration of biotinylated Cynomolgus ACVR1 is 1300 µg/mL.
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 Rat CD157: 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 Rat CD157: 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 Rat CD157 protein concentration as the horizontal axis, and calculate the ED₅₀ value.

Rat

HEK293

C-6*His

Q63072 (R34-E293)

81506  [NCBI]

Partial

RWSGEGTTPHLQSIFLGRCAEYTTLLSLEPGNKNCTAIWEAFKVVLDKDPCSVLPSDYDLFINLSRHAIPRDKSLFWENNHLLVMSYAENTRRLMPLCDVLYGKVGDFLSWCRQENASGLDYQSCPTAEDCENNAVDAYWKSASMQYSRDSSGVINVMLNGSEPKGAYPTKGFFADFEIPYLQKDKITRIEIWVMHEVGGPHVESCGEGSVKILEDRLEALGFQHSCINDYPPVKFLMCVDHSTHPDCAMNSASASMWRE

Approximately 33-45 kDa due to the glycosylation.

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  Greater than 95% 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.