3. Prepare a 200 mM ammonium acetate solution pH 6.9 by
weighting 770.8 mg in 50 mL.
4. Reconstitute GroEL (Chaperonin 60 from Escherichia coli,
Sigma-Aldrich reference no. C7688) powder (1 mg) by adding
80 μL of refolding buffer and 20 μL of methanol directly into
the vial.
5. Shake for 2 h at room temperature.
6. Transfer the entire volume into an Eppendorf, add 100 μL
acetone, and agitate gently.
7. After 2 min, centrifuge for 1 min at 11,000 Â g: a white
precipitate should be present. Delete the supernatant. Then,
add 200 μL of refolding buffer and agitate gently until complete dissolution.
8. Shake for 1 h at room temperature. Centrifuge for 1 min at
1,500 Â g and take the supernatant to perform the first desalting step as explained in Subheading 3.1 (on Zeba™ spin
column).
9. The second desalting step is performed as explained in Subheading 3.2 with 10 cycles (each one takes between 8 and
15 min) at 1.9 Â g with a 50-kD MWCO membrane. Finally,
check the protein concentration by UV absorbance at 280 nm.
Typically, approximately 10 μM of 14-mer should be obtained
(ε ¼ 10,430 M
À1 cm
À1 for GroEL monomer at 280 nm).
3 Methods
3.1 Benchtop Size
Exclusion
Chromatography
Desalting
1. Operate at +4
C.
2. You can use 6 K MWCO Micro Bio-Spin™ 6 (Biorad) or
0.5 mL 7 K MWCO Zeba™ (Thermo Fisher Scientific) spin
columns.
3. Remove the storage solution by centrifugation for 2 min at
1,000 Â g for Micro Bio-Spin™ columns and 1 min at
1,500 Â g for Zeba™ columns.
4. Equilibrate the columns by adding 500 μL (Micro Bio-Spin™)
or 300 μL (Zeba™) of 200 mM ammonium acetate solution,
centrifuge for 1 min at 1,000 Â g (Micro Bio-Spin™) or at
1,500 Â g (Zeba™) and discard the eluate.
5. Repeat step 3 three times.
6. Load between 20 and 75 μL (Micro Bio-Spin™) or 30 and
130 μL (Zeba™) of sample. Loading smaller volume may affect
recovery (see Note 3).
Native Mass Spectrometry
175
weighting 770.8 mg in 50 mL.
4. Reconstitute GroEL (Chaperonin 60 from Escherichia coli,
Sigma-Aldrich reference no. C7688) powder (1 mg) by adding
80 μL of refolding buffer and 20 μL of methanol directly into
the vial.
5. Shake for 2 h at room temperature.
6. Transfer the entire volume into an Eppendorf, add 100 μL
acetone, and agitate gently.
7. After 2 min, centrifuge for 1 min at 11,000 Â g: a white
precipitate should be present. Delete the supernatant. Then,
add 200 μL of refolding buffer and agitate gently until complete dissolution.
8. Shake for 1 h at room temperature. Centrifuge for 1 min at
1,500 Â g and take the supernatant to perform the first desalting step as explained in Subheading 3.1 (on Zeba™ spin
column).
9. The second desalting step is performed as explained in Subheading 3.2 with 10 cycles (each one takes between 8 and
15 min) at 1.9 Â g with a 50-kD MWCO membrane. Finally,
check the protein concentration by UV absorbance at 280 nm.
Typically, approximately 10 μM of 14-mer should be obtained
(ε ¼ 10,430 M
À1 cm
À1 for GroEL monomer at 280 nm).
3 Methods
3.1 Benchtop Size
Exclusion
Chromatography
Desalting
1. Operate at +4
C.
2. You can use 6 K MWCO Micro Bio-Spin™ 6 (Biorad) or
0.5 mL 7 K MWCO Zeba™ (Thermo Fisher Scientific) spin
columns.
3. Remove the storage solution by centrifugation for 2 min at
1,000 Â g for Micro Bio-Spin™ columns and 1 min at
1,500 Â g for Zeba™ columns.
4. Equilibrate the columns by adding 500 μL (Micro Bio-Spin™)
or 300 μL (Zeba™) of 200 mM ammonium acetate solution,
centrifuge for 1 min at 1,000 Â g (Micro Bio-Spin™) or at
1,500 Â g (Zeba™) and discard the eluate.
5. Repeat step 3 three times.
6. Load between 20 and 75 μL (Micro Bio-Spin™) or 30 and
130 μL (Zeba™) of sample. Loading smaller volume may affect
recovery (see Note 3).
Native Mass Spectrometry
175