Cathepsin B 蛋白, Mouse (HEK293, His)

Cathepsin B Protein, Mouse (HEK293, His) is an approximately 32-50 kDa Cathepsin B protein with a His-flag. Cathepsin B is an enzymatic protein belonging to the peptidase C1 family^[1].

组织蛋白酶 B (CTSB) 也称为 APP 分泌酶 (APPS) 和 CPSB，是属于肽酶 C1 家族的酶蛋白^[1]。
组织蛋白酶 B/CTSB 是作为前酶原，并在递送至溶酶体之前在前溶酶体酸性囊泡（晚期内涵体）中被激活。去除信号肽后，无活性的酶原会经历修饰，包括去除原区域，从而产生活性酶^[1]。
包括组织蛋白酶 B 在内的几种溶酶体蛋白酶与肿瘤的恶性进展。许多研究人员已经证明组织蛋白酶 B 活性增强与动物肿瘤或人类肿瘤的转移能力增强之间存在相关性^[2]。

Measured by its ability to cleave 10μM fluorogenic peptide 10μM substrate Z-LR-AMC. The specific activity is 43654.4-47491.133 pmol/min/µg, as measured under the described conditions. (Activation description: The proenzyme needs to be activated in acid reducing buffer for an activated form.)

Materials
Activation Buffer: 25 mM MES, 5 mM DTT, pH 5.0.
Assay Buffer: 25 mM MES, pH 5.0.
Cathepsin B Protein, Mouse (HEK293, His) (HY-P7747)
Substrate: 7-Amino, 4-Methyl Coumarin (AMC, HY-D0027)

Experimental steps
1. Standard curve: Dilute 7-Amino, 4-Methyl Coumarin (AMC) (standard) to 0, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 μmol/L with assay buffer, and add 100 μL into the black enzyme-labeled wells. The excitation and emission wavelengths were 380 nm and 460 nm, respectively, and the standard curve was made with the measured RFU as the horizontal coordinate and the amount of standard substance as the vertical coordinate to obtain the standard curve formula.
2. Dilute the measured proteins to 10μg/mL with activation buffer.
3. Incubate for 15 minutes at room temperature.
4. Dilute Mouse Cathepsin B to 0.2 μg/mL in Assay Buffer.
5. Dilute substrate to 20 μM in assay buffer.
6. Add 50 μL of each concentration of the measured protein to the assay wells and add 50 μL of 20 μM substrate to start the reaction. Blank wells were spiked with 50 μL of assay buffer and 50 μL of 20μM protein-free substrate.
7. The excitation and emission wavelengths were 380 nm and 460 nm, respectively, and read in kinetic mode for 5 minutes.
8. Calculate the specific activity:

Mouse

HEK293

C-6*His

P10605 (H18-F339)

13030  [NCBI]

Full Length of Mature Protein

HDKPSFHPLSDDLINYINKQNTTWQAGRNFYNVDISYLKKLCGTVLGGPKLPGRVAFGEDIDLPETFDAREQWSNCPTIGQIRDQGSCGSCWAFGAVEAISDRTCIHTNGRVNVEVSAEDLLTCCGIQCGDGCNGGYPSGAWSFWTKKGLVSGGVYNSHVGCLPYTIPPCEHHVNGSRPPCTGEGDTPRCNKSCEAGYSPSYKEDKHFGYTSYSVSNSVKEIMAEIYKNGPVEGAFTVFSDFLTYKSGVYKHEAGDMMGGHAIRILGWGVENGVPYWLAANSWNLDWGDNGFFKILRGENHCGIESEIVAGIPRTDQYWGRFHHHHHH

32-50 kDa

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

无菌粉末

Lyophilized after extensive dialysis against PBS, pH7.4.

<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.

• [1]. Izabela Podgorski, et al. Cathepsin B and its role(s) in cancer progression. Biochem Soc Symp. 2003;(70):263-76.  [Content Brief]

  [2]. W Morikawa, et al. Angiostatin generation by cathepsin D secreted by human prostate carcinoma cells. J Biol Chem. 2000 Dec 8;275(49):38912-20.  [Content Brief]