ADAM8 Protein, Rhesus Macaque (HEK293, His)

ADAM8 is a transmembrane glycoprotein containing metalloproteinase and disintegrin domains, belonging to the ADAMs family. It has proteolytic, cell adhesion, cell fusion, and cell signaling properties. It is involved in ectodomain shedding of membrane proteins and is associated with inflammation and neurodegeneration. ADAM8 Protein, Rhesus Macaque (HEK293, His) is the recombinant Rhesus Macaque-derived ADAM8 protein, expressed by HEK293 , with C-His labeled tag.

In response to inflammatory stimuli such as lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α), ADAM8 expression is upregulated in macrophages and activated glial cells (astrocytes and microglia) in the central nervous system (CNS), suggesting its involvement in neuronal-glial signaling. ADAM8 has proteolytic, cell adhesion, cell fusion, and cell signaling properties. It is involved in ectodomain shedding of membrane proteins and is associated with inflammation and neurodegeneration^[1][2].

Measured by its ability to cleave a fluorogenic peptide substrate Mca-PLAQAV-Dpa-RSSSR-NH₂. The specific activity is 0.77 pmol/min/μg, as measured under the described conditions. (Activation description: The proenzyme needs to be activated by Thermolysin for an activated form.)

Materials
Assay buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5 (TCN)
ADAM8 Protein, Rhesus Macaque (HEK293, His) (HY-P772930)
Bacterial Thermolysin
Phosphoramidon: Dissolve in methanol to a concentration of 20 mM Substrate: Mca-PLAQAV-Dpa-RSSSR-NH2 (HY-P3722A)
Standard: MCA-Pro-Leu-OH

Procedure
1. Standard curve preparation: Dilute MCA-Pro-Leu-OH (standard) in assay buffer to the following concentrations: 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78, 0.39, 0.19, 0 pmoL. Add 100 μL of each standard concentration to a 96-well plate. Read in kinetic mode for 5 minutes at excitation and emission wavelengths of 320 nm and 405 nm, respectively. Plot the standard concentration on the x-axis and the measured RFU on the y-axis to generate a standard curve and obtain the standard curve equation.
2. Dilute the test protein to 400 μg/mL in assay buffer.
3. Mix equal volumes of 1.5 μg/mL Thermolysin and diluted test protein.
4. Incubate at 37°C for 30 minutes.
5. Add Phosphoramidon to a final concentration of 0.05 mM to stop the reaction.
6. Incubate at room temperature for 15 minutes.
7. Dilute the activated test protein to 40 ng/μL in assay buffer.
8. Dilute the substrate to 40 μM in assay buffer.
9. Load 50 μL of 40 ng/μL test protein into a black plate and add 50 μL of 40 μM substrate. For the blank, add 50 μL of assay buffer and 50 μL of 40 μM substrate.
10. Read in kinetic mode for 5 minutes at excitation and emission wavelengths of 320 nm and 405 nm, respectively.
11. Calculate the specific activity:

Rhesus Macaque

HEK293

C-His

XP_002805919 (R21-P497)

100428452  [NCBI]

Partial

RPWARVERYEVVLPRRLPGPRVRRALPSHVGLYPERVSYVLGATGHNFTLHLRKNRDLLGSGYTETYTAANGSEVTEQPRGQDHCFYQGHVEGHPDSAASLSTCAGLRGFFQVGSDLHLIEPLDEGGEGGRHAVYEAEHLLQTAGTCGVSDDSLGSLLGPRTAAVFRPQPGGSLPSRETRYVELYVVADNAEFQMLGSEAAVRHRVLEVVNHVDKLYQKLNFRVVLVGLEIWNRQDRFHVSPHPDVTLENLLAWQAQRLTQRHLQDNVQLITGVDFTGTTVGLARVSAMCSHGSGAVNQDHSKNPVGVACTMAHEMGHNLGMDHDENVQGCHCREPTEAGRCIMAGSIGSTFPRMFSDCSRAYLEGFLEQPQSACLANAPDLSHLVGGPVCGNLFVERGEQCDCGPPEDCRNHCCNSTTCQLAEGAQCAHGTCCQECRVKPAGELCRPKKDTCDLEEFCDGRHPECPEDAFQENGTP

55-60kDa.

• 
  Greater than 95% as determined by reducing SDS-PAGE.

Solution.

Supplied as a 0.22 μm filtered solution of 12 mM Tris-HCl, 5 mM CaCl₂, 75 mM NaCl, pH 7.4, 50% glycerol.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

N/A.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.