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Amylose Affinity Chromatography of MBP 181
9. Resuspend in 10 RV of equilibration buffer, thoroughly vortex and incubate on
ice for 30 min.
10. Pellet the matrix and decant the supernatant as in step 7.
11. Carefully add the clarified (bacterial cell lysate) to the matrix and gently mix to
a suspension (see Note 12).
12. Incubate the suspension for 1hat4°Conasuitable shaker (e.g., rocking or
platform) at a low speed setting (see Note 13).
13. Pellet the matrix and decant the supernatant as in step 7. Retain the supernatant
as required in a separate vessel for analysis of the unbound flow-through.
14. Carefully add 5 RV of wash buffer 2, gently mix and transfer to a fresh vessel
(see Note 17).
15. Pellet the matrix and decant the supernatant as in step 7.
16. Continue to washing using 5 RV wash buffer 2 until the A 280 nm is stable between
0.01 and 0.001 blanking with wash buffer 2.
17. Pellet the matrix and decant the supernatant as in step 7.
18. Resuspend the matrix in 1 RV of wash buffer 2 and either transfer to a smaller
vessel for elution (proceed to step 19), or load to a column (proceed to step 21).
19. Pellet the matrix and decant the supernatant as in step 7 and elute the MBPpassenger
protein complex from the matrix by adding 1 RV of elution buffer (see
Note 14) and resuspend gently with a pipette and incubate for 10 min on ice.
20. Centrifuge at 4000 × g for 5 min, collecting the supernatant containing the
MBP-passenger protein. Regenerate the matrix using the steps described in
Subheading 3.5.
21. Wash the column with 1 RV of wash buffer 2 and elute the MBP-passenger
protein by adding 0.5–1 mL aliquots of elution buffer (see Note 14) and allowing
it to enter the matrix, collecting similar sized fractions.
22. Check the A 280 nm to identify fractions containing the desired MBP-passenger
protein.
23. Regenerate the matrix using the steps described in Subheading 3.5.
3.4. FPLC Purification: Amylose High Flow Affinity Chromatography
1. Measure the total protein concentration of the cleared lysate from the calculation
experiment (see Subheading 3.2.).
2. Calculate the amount of matrix for purification and pour column.
3. Attach the column to the FPLC, and wash with 5 column volumes (CV) of column
preparation solution under operational conditions (see Note 2).
4. Wash once with 5 CV of pre-equilibration buffer.
5. Wash with 10 RV of column buffer. Confirm equilibration by measuring pH and
conductivity. Continue equilibration until pH and conductivity from the column
matches equilibration buffer.
6. Load the bacterial cell lysate (see Note 11) at 2.5 mg/mL protein concentration
onto the column in accord with conditions given in Note 2.
7. Wash with 10 CV of column buffer.