228
- Clear additional cell debris via centrifugation at 800 × g for
1 min at 4 °C. - Transfer supernatant to chilled new microcentrifuge tube.
This is the total cellular protein extract. The extract may be
split into aliquots and stored in −80 °C. - Quantify total protein extract and take equal amount of pro-
tein from all samples. - Normalize volume with buffer B containing 0.5 % (v/v) prote-
ase inhibitors. - Sediment protein by centrifugation at 15,000 × g for 15 min
at 4 °C. - Transfer supernatant to separate microcentrifuge tube. This
fraction contains soluble proteins. Store on ice for the remain-
der of the procedure. The remaining pellet fraction contains
insoluble proteins. - Add 400 μL of wash buffer to the tube containing the protein
pellet. - Vortex at setting 3 for 10 s.
- Centrifuge at 15,000 × g for 15 min to collect the protein
pellet. - Discard supernatant and repeat steps 5 – 7.
- Add original amount of buffer B containing 0.5 % (v/v) prote-
ase inhibitors to the protein pellet. - Vortex sample at 4 °C to completely dissolve the pellet.
- The resulting fractions may be analyzed via SDS-PAGE or
Filter Trap Assay. - Prepare protein fractions as described under Subheading 3.3
starting with 2,000 μg of total protein lysate. Adjust volume to
set concentration to 5 μg/μL. - Serially dilute both of the fractions for each experimental sam-
ple: Start with 75 μL of the sample plus 125 μL buffer B con-
taining 0.5 % (v/v) protease inhibitors. Mix well by gentle
vortex (setting 3) for 10 s. Label this mixture as D1. - For dilution 2 (D2), take 100 μL of D1 and add that to 100 μL
of buffer B containing 0.5 % (v/v) protease inhibitors. Mix by
gentle vortex for 10 s. - For dilution 3 (D3), take 100 μL of D2 and add that to 100 μL
of buffer B containing 0.5 % (v/v) protease inhibitors. Mix by
gentle vortex for 10 s. - For dilution 4 (D4), take 100 μL of D3 and add that to 100 μL
of buffer B containing 0.5 % (v/v) protease inhibitors. Mix by
gentle vortex for 10 s. Discard 100 μL.
3.3 Sedimentation
Assay
3.4 Filter Trap Assay
Amit Shrestha et al.http://www.ebook3000.com