ADAM17/TACE Protein, Rat (HEK293, His)

ADAM17/TACE Protein, a pivotal enzyme, cleaves TNF-alpha and activates various cell-surface proteins like p75 TNF-receptor. It also mediates the cleavage of Notch, activates the Notch pathway, and contributes to hemostasis by shedding GP1BA. ADAM17/TACE further facilitates the cleavage of LAG3, IL6R, and FCGR3A, showcasing its significance in diverse cellular functions. ADAM17/TACE Protein, Rat (HEK293, His) is the recombinant rat-derived ADAM17/TACE protein, expressed by HEK293 , with C-10*His labeled tag.

ADAM17, also known as TACE, serves as a pivotal enzyme in diverse cellular processes, notably cleaving the membrane-bound precursor of TNF-alpha to its mature soluble form. Beyond its role in TNF-alpha processing, ADAM17 exhibits a broad substrate specificity, orchestrating the proteolytic release of various cell-surface proteins such as p75 TNF-receptor, interleukin 1 receptor type II, p55 TNF-receptor, transforming growth factor-alpha, L-selectin, growth hormone receptor, MUC1, and the amyloid precursor protein. Moreover, ADAM17 acts as an activator of the Notch pathway by mediating the cleavage of Notch, generating the membrane-associated intermediate fragment known as Notch extracellular truncation (NEXT). Its involvement extends to hemostasis through shedding the platelet glycoprotein Ib alpha chain (GP1BA) and mediating the cleavage of LAG3, IL6R, and FCGR3A, highlighting its significance in diverse cellular functions.

Measured by its ability to cleave a fluorogenic peptide substrate Mca-PLAQAV-Dpa-RSSSR-NH2 and the specific activity is 250-2200 pmol/min/µg.

Materials
Assay buffer: 25 mM Tris, 2.5 μM ZnCl₂, 0.005% (w/v) Brij-35, pH 9.0 (TCNB)
ADAM17/TACE Protein, Rat (HEK293, His) (HY-P72815)
Substrate: Mca-PLAQAV-Dpa-RSSSR-NH₂ TFA (HY-P3722A), dissolved in 2 mM DMSO
Standard: MCA-Pro-Leu-OH

Procedure
1.Standard Preparation: Dilute the standard in assay buffer to concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0 μmol/L. Add 100 μL of each concentration to the wells and measure the RFU at excitation and emission wavelengths of 320 nm and 405 nm, respectively.
2. Dilute recombinant Rat TACE/ADAM17 protein in assay buffer to concentrations of 0.02 ng/mL, 0.2 ng/mL, 2 ng/mL.
3. Dilute the substrate to 20 μM in assay buffer.
4. Experimental group: Mix 50 μL of different concentrations of recombinant Rat TACE/ADAM17 protein with 50 μL of 20 μM substrate.
Control group: Mix 50 μL of assay buffer with 50 μL of 20 μM substrate.
5. Read the fluorescence in kinetic mode for 5 minutes at excitation and emission wavelengths of 320 nm and 405 nm, respectively.
6. Calculate enzyme activity:

Rat

HEK293

C-10*His

Q9Z1K9 (P18-D563)

57027  [NCBI]

Partial

PRPPEEPGSGSHLRLEKLDSLLSDYDILSLSNIQQHSIRKRDLQSATHLETLLTFSALKRHFKLYLTSSTERFSQNLRVVVVDGKEESEYSVKWQDFFSGHVVGEPDSRVLAHIGDDDVTVRINTDGAEYNIEPLWRFVNDTKDKRMLVYKSEDIKDFSRLQSPKVCGYLNADSEELLPKGLIDREPSEEFVRRVKRRAEPNPLKNTCKLLVVADHRFYKYMGRGEESTTTNYLIELIDRVDDIYRNTSWDNAGFKGYGVQIEQIRILKSPQEVKPGERHFNMAKSFPNEEKDAWDVKMLLEQFSLDIAEEASKVCLAHLFTYQDFDMGTLGLAYVGSPRANSHGGVCPKAYYNPGVKKNIYLNSGLTSTKNYGKTILTKEADLVTTHELGHNFGAEHDPDGLAECAPNEDQGGKYVMYPIAVSGDHENNKMFSNCSKQSIYKTIESKAQECFQERSNKVCGNSRVDEGEECDPGIMYLNNDTCCNSDCTLKPGVQCSDRNSPCCKNCQFETAQKKCQEAINATCKGVSYCTGNSSECPPPGDAED

Approximatel 57 (Mature form)&20 kDa (Propeptide), on SDS-PAGE under reducing conditions.

• 
  ≥ 90%, as determined by reducing SDS-PAGE.

Lyophilized powder

1.Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4, 5% trehalose, 5% mannitol, 0.01% Tween 80.
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.