5. Measure protein concentration to make sure it is equal in each
sample.
3.8 Affinity
Purification with
StreptavidinSepharose Beads
1. Prepping the streptavidin Sepharose beads
(a) Thoroughly mix the streptavidin Sepharose beads
slurry mix.
(b) For “N” number of samples transfer a total of
(N + 2) Â 30 μL of slurry into a fresh 1.5 mL
microcentrifuge tube.
(c) Wash slurry with 1 mL of protein extraction buffer (no detergents) by inversion 4–5 times.
(d) Centrifuge at 500 Â g for 1 min.
(e) Remove supernatant being careful not to disturb the
beads. Repeat steps 1c–e three more times.
(f) Resuspend beads in (N + 2) Â 15 μL of protein extraction
buffer (no detergents) after final wash.
2. Add 30 μL of washed streptavidin Sepharose beads to each
sample from Subheading 3.7, step 4 after equalizing all the
protein concentrations for each sample.
3. Rotate samples with beads overnight at 4
C on a rotator (see
Note 26).
4. Next day, centrifuge beads for 2 min at 500 Â g.
5. Remove supernatant without disturbing beads (see Notes
27–31).
6. Add 1 mL of protein extraction buffer (no detergents) to the
beads (see Note 32).
7. Transfer solution with beads to a fresh non-autoclaved 1.5 mL
microcentrifuge tube.
8. Centrifuge solution with beads for 30 s at 500 Â g. Remove
supernatant.
9. Wash beads with 1 mL of 2% wash buffer (see Subheading 2.9,
step 4).
10. Centrifuge for 30 s at 500 Â g. Remove supernatant.
11. Wash beads with 1 mL of protein extraction buffer
(no detergents).
12. Centrifuge for 30 s at 500 Â g. Remove supernatant (repeat
steps 11 and 12 twice).
13. Wash beads with 1 mL of 50 mM ABC.
14. Centrifuge for 30 s at 500 Â g. Remove supernatant (repeat
steps 13 and 14 three times. Leave the beads in the final ABC
wash while preparing the trypsin solution).
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Madiha Khan et al.
sample.
3.8 Affinity
Purification with
StreptavidinSepharose Beads
1. Prepping the streptavidin Sepharose beads
(a) Thoroughly mix the streptavidin Sepharose beads
slurry mix.
(b) For “N” number of samples transfer a total of
(N + 2) Â 30 μL of slurry into a fresh 1.5 mL
microcentrifuge tube.
(c) Wash slurry with 1 mL of protein extraction buffer (no detergents) by inversion 4–5 times.
(d) Centrifuge at 500 Â g for 1 min.
(e) Remove supernatant being careful not to disturb the
beads. Repeat steps 1c–e three more times.
(f) Resuspend beads in (N + 2) Â 15 μL of protein extraction
buffer (no detergents) after final wash.
2. Add 30 μL of washed streptavidin Sepharose beads to each
sample from Subheading 3.7, step 4 after equalizing all the
protein concentrations for each sample.
3. Rotate samples with beads overnight at 4
C on a rotator (see
Note 26).
4. Next day, centrifuge beads for 2 min at 500 Â g.
5. Remove supernatant without disturbing beads (see Notes
27–31).
6. Add 1 mL of protein extraction buffer (no detergents) to the
beads (see Note 32).
7. Transfer solution with beads to a fresh non-autoclaved 1.5 mL
microcentrifuge tube.
8. Centrifuge solution with beads for 30 s at 500 Â g. Remove
supernatant.
9. Wash beads with 1 mL of 2% wash buffer (see Subheading 2.9,
step 4).
10. Centrifuge for 30 s at 500 Â g. Remove supernatant.
11. Wash beads with 1 mL of protein extraction buffer
(no detergents).
12. Centrifuge for 30 s at 500 Â g. Remove supernatant (repeat
steps 11 and 12 twice).
13. Wash beads with 1 mL of 50 mM ABC.
14. Centrifuge for 30 s at 500 Â g. Remove supernatant (repeat
steps 13 and 14 three times. Leave the beads in the final ABC
wash while preparing the trypsin solution).
436
Madiha Khan et al.