Tissue Extract Protocols for PAI-1 Activity Kits

Sample Tissue Extract Protocols for PAI-1 Activity Kits

Simple Protocol
1. Homogenize tissue samples on ice in cold lysis buffer.
2. Centrifuge samples at 4C and remove supernates.
3. Snap freeze samples on dry ice and store at -70C.

Lysis buffer
0.05 M Sodium Phosphate pH 6.6
0.15 M Sodium Chloride
0.01 M EDTA
0.5% Triton X-100

Dialysis protocol
1. Homogenize tissue samples on ice in cold lysis buffer.
2. Centrifuge samples at 4C and remove supernates.
3. Dialyze supernates against dialysis buffer for 2-4 hours at 4C.
4. Snap freeze samples on dry ice and store at -70C.

Lysis buffer
0.05 M Sodium Phosphate pH 6.6
0.15 M Sodium Chloride
0.01 M EDTA
0.01 M Benzamidine
0.01 M DTT
0.005 M PMSF
0.02% Tween 20

Dialysis buffer
0.05 M Sodium Phosphate pH 6.6
0.15 M Sodium Chloride
0.01 M EDTA

Some common components in general tissue extract protocols may interfere with PAI-1 activity and the function of the assay. Detergent will cause active PAI-1 to become latent and inactive, so concentrations and sample contact time should be minimized. Protease inhibitors should be avoided in the final buffer because they will interfere with the assay. PAI-1 activity may not be completely preserved, so results from the assay should be considered relative and not absolute.

The above procedures are intended for use as a reference. Optimal concentrations, experimental conditions and experimental processes are to be determined by the individual user. No guarantee of performance using the above procedure is expressed or implied.

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