Activity Measured by its ability to be used as a protein substrate for TACE/ADAM17.
Under the described conditions TACE/ADAM17 will cleave pro-TNF-a to produce mature TNF-a.
Assay Procedure
Materials:
Assay Buffer: 50 mM Tris, pH 8.0
Recombinant Human Pro TNF-a Fusion Protein (rhPro-TNF-a) (Catalog # 1012-PS)
Recombinant Human TACE/ADAM17 (rhTACE) (Catalog # 930-ADB)
Positive Control: Recombinant Human TNF-a (rhTNF-a) (Catalog # 210-TA)
Goat Anti-Human TNF-a Biotinylated Antigen Affinity-purified Polyclonal Antibody (Catalog # BAF210)
SDS-PAGE followed by Western Blotting
Dilute rhPro-TNF-a to 0.2 mg/mL in Assay Buffer.
Dilute rhTACE to 0.2 mg/mL in Assay Buffer.
Prepare the following vials for a final volume of 20 uL.
10 uL of rhPro-TNF-a at 0.2 mg/mL + 10 uL of rhTACE at 0.2 mg/mL
10 uL of rhPro-TNF-a at 0.2 mg/mL + 10 uL of Assay Buffer (control-with incubation)
10 uL of rhPro-TNF-a at 0.2 mg/mL + 10 uL of Assay Buffer (control-without incubation)
Note: At this point the concentration of rhPro-TNF-a in the reaction tubes is 0.1 mg/mL.
Incubate vials at 37C overnight (except for the non-incubated control, store at =-20C).
Stop the reactions by adding the reducing gel loading buffer for SDS-PAGE to all vials. Heat the samples at 100C for 3-5 minutes.
Dilute the reaction tubes (and control without incubation) to 50 ng/15 uL in reducing sample buffer.
Prepare a sample of Positive Control at 5 ng/15 uL in reducing sample buffer. Heat the sample at 100C for 3-5 minutes.
Load the samples on a 15% gel.
50 ng/lane (15 uL) of rhPro-TNF-a of the incubated reactions (including the control with incubation).
50 ng/lane (15 uL) of rhPro-TNF-a of the control (without incubation).
5 ng/lane (15 uL) of Positive Control (Catalog # 210-TA).
Follow SDS-PAGE/Western Blotting procedures.
Use the Biotinylated Anti-human TNF-a/TNFSF1A antibody at 0.1 ug/mL. (Catalog # AF-210-NA may be used in place of # BAF210.)
Visually determine processing of rhPro-TNF-a to the mature form by rhTACE by comparing the incubated reactions to the Positive Control.
