Activity assay of trypsin solution and the inhibition of aprotinin

Objective:

1. Determine activity of trypsin-EDTA solution for cell culture from Sigma.

2. Determine the amount ratio of recombinant aprotinin to trypsin activity.

Materials：

1) Trypsin-EDTA solution , sigma T4049-100ml  Lot # SLBN6048V  EXP Aug 17, 0.25%, sterile filtered, BioReagent, suitable for cell culture, 2.5g porcine trypsin and 0.2g EDTA per liter

Hank’s Balanced Salt Solution with phenol red.

2) RTI (recombinant aprotinin) Lot # 160301 , 5.8 EPU/mg, 21.9mg/ml, Shanghai Yaxin Biotechnology Co. Ltd.

3) Activity assay substrate：BAEE solution，preparing of solution according to company SOP.

4) Trypsin activity assay: based on YaxinBio SOP.

Method and Results：

1) Determine activity of trypsin-EDTA solution：4150 BAEE unit/ml.

2) 1EPU RTI can inhibit the 16200 BAEE unit trypsin activity .

Completely inhibit 4150 BAEE unit / ml trypsin and the usage amount of RTI is 4150/16200 = 0.256 EPU.

Detailed experiment is as follow:

2.1 Take 150ul trypsin-EDTA solution, the trypsin activity is 622.5 BAEE unit (4150 BAEE unit / ml * 0.15).

2.2 The RTI volume is calculated as follow: 622.5 / 16200 = 0.0384 EPU, 0.0384 / (5.8 * 21.9) = 0.302ul, and then the RTI is diluted for 10 times, taking 3.02 ul to use.

After 5min reaction , trypsin activity was measured.

Result: no activity measured, trypsin was completely inhibited.

2.3 Determine the RTI inhibition rate if add in half amount, 1.51 µl (which is half of 3.02µl). Other operation is the same as 2.2.

After 5min reaction , trypsin activity was measured.

Trypsin activity : 2100BAEE unit / ml.

Result: The inhibition rate is 49.39%.

Conclusion：

1) 1EPU RTI completely inhibit 16200 BAEE unit trypsin activity , if not completely inhibited, the inhibition rate proportional to the amount of RTI.

2) Confirm the RTI amount in accordance with the trypsin activity.

3) Trypsin-EDTA solution, sigma T4049-100ml Lot # SLBN6048V, the activity is 4150 BAEE / ml,

If completely inhibited, 0.256 EPU aprotinin was added per ml.

If add half amount of aprotinin , half trypsin activity was inhibited.