Description
Trypsin hydrolyzes proteins, peptides, amides and esters specifically at the carboxyl groups of the basic amino acids L-arginine or L-lysine.
F7m: 1. 0 mg trypsin per CR-column, immobilized on polyvinyl
10,200 ST-units immobilized per CR-column.
Nr. 15 Storage buffer: 50 mM Tris/HCl at pH 8. 0 at 4°C
Nr. 67 Reaction buffer: 50 mM phosphate at pH 8. 0
Nr. 68 Washing buffer: 50 mM phosphate at pH 8. 0, 1 M NaCl
Enzyme Commission Number
EC 3. 4. 21. 4
Synonyms
α-trypsin; β-trypsin; cocoonase; parenzyme; parenzymol; tryptar; trypure; pseudotrypsin; tryptase; tripcellim; sperm receptor hydrolase; Alpha-trypsin; Beta-trypsin; EC 3. 4. 21. 4; Trypsin
Protocol
1. Dilute delivered buffers (at least 2 ml each) with doubly distilled water.
For 1 application you need:
0.25 ml 10x reaction buffer and 2. 25 ml doubly distilled water
0.4 ml 5x washing buffer and 1. 6 ml doubly distilled water
0.2 ml 10x storage buffer and 1. 8 ml doubly distilled water
The substrate should be in reaction buffer
2. Equilibrate the CR-column with 2 ml reaction buffer.
Load in a syringe 2 ml reaction buffer, let the reaction buffer run through the column by gravity to the upper filter. In case the buffer runs very slowly, apply pressure by a syringe.
3. Load substrate solution in reaction buffer.
Small volumes (< 80 µl): spin the CR-column 5 seconds in a benchtop centrifuge (2,000 rpm are sufficient). Let the substrate solution enter the matrix material.
Larger volumes: Let the substrate solution run through the column.
Flow-rate: up to 80 µl/minute
Keep the substrate in the column for about 1 minute at room temperature. Higher turn-over is obtained when the substrate is applied to the column again or incubated for longer times.
4. Elute the product solution.
Small volumes (< 80 µl): Elute the product with 500 µl reaction buffer.
Larger volumes: Let the substrate run through the column and elute the residual product solution with 500 µl reaction buffer.
It does not harm the columns if they run dry.
5. Wash the column with 2 ml washing buffer.
6. Equilibrate the column with 2 ml storage buffer.
Store the column at 4°C.
Never freeze a CR-column!
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