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21 Nov 2009

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	Tissue Extract Protocol for PAI-1 Activity Kits

Solution Sample Tissue Extract Protocol for PAI-1 Activity Kits

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 procedure is 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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16 Jan 2009 05:14 AM