Bio-Rad Human MMP and TIMP Assays - Manual (Page 18)

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5. Perform either of the following to remove insoluble cellular particulates:
n
Centrifuge the cell lysate solution at 4,500 x g for 20 min at 4°C,
and then filter the lysate using a 0.45 μm syringe filter
n
If the lysate volume is not adequate for filtration, centrifuge the lysate
at 15,000 x g for 10 min at 4°C using a benchtop microcentrifuge
6. Collect the filtered lysate or supernatant after centrifugation.
7. Measure protein concentration using Bio-Rad’s DC ™ protein assay
kit and if needed, adjust protein concentration with cell lysis buffer
containing PMSF and cell lysis factor QG.
8. The suggested working protein concentration range for these assays
is 200–900 μg/ml (10-45 µg per assay well).
9. Store the aliquoted lysates at –70°C until ready to use
Suspension Cells
1.
Collect cell suspension and pellet the cells by spinning at 1,000 x g for
5 min at 4°C.
2. Aspirate off cell culture medium completely.
3. Wash by resuspending the cells with ice-cold cell wash buffer (bottle
with the blue cap) .
4. Centrifuge the cells at 1,000 x g for 5 min at 4ºC.
5. Completely remove the buffer.
6. Immediately add the proper volume of cell lysis buffer and gently rock for
20 min at 4°C.
7. Remove insoluble cellular particulates as described in Adherent Cells step
5 above .
8. Follow Adherent Cells steps 6–9 above.
Tissue Samples
1. Cut the tissue into small pieces (~3 x 3 mm) for ease of handling and
blood removal. If necessary, wash the tissue with ice-cold cell wash
buffer (bottle with the blue cap) to completely remove all blood. Then
transfer the tissue to a 2 ml tissue grinder.