2 Materials
2.1 Protein Samples
1. BSA (SIGMA) was dissolved at 5 mg/mL in 20 mM Na-K
Phos 150 mM NaCl pH 7 and filtered at 45 μm. BSA1 sample
was obtained by five times dilution to 1 mg/mL. BSA2 sample
at 0.8 mg/mL was obtained from the main elution peak of
500 μL BSA stock sample injected at 0.5 mL/min on a Superose 12 10/300 GL (GE Healthcare) size exclusion chromatography column.
2. Recombinant BTG2 fused to glutathione S transferase
(GST-BTG2) and PAPB proteins were purified by affinity and
size exclusion chromatography [20]. Stocks of GST-BTG2
protein at 1.1 mg/mL and PABP at 5.1 mg/mL in buffer
10 mM K phosphate, pH 7.5, 100 mM NaCl, 0.5 mM TCEP
were used in this study (see Note 3). In the dilution series,
mixtures contain a fixed concentration of GST-BTG2 (5 μM)
and a range of seven PABP concentrations to give molar ratio
(PABP/GST-BTG2) of 1:2, 1:1, 2:1, 4:1, 8:1, 16:1, and 32:1.
2.2 Instrument
and Sample Cells
1. An analytical ultracentrifuge (Optima XLI Beckman) and rotor
(8-hole AnTi-50, Beckman). Analytical ultracentrifugation cell
assemblies equipped with sapphire windows and two-channel
Titanium, Aluminum or Epon centerpieces of 1.5, 3, and/or
12 mm optical path length (Nanolytics or Beckman).
2. Programs: The program SEDNTERP created by D. Hayes,
T. Laue, J. Philo and available free (http://bitcwiki.sr.unh.
edu/index.php/Downloads) for calculating the parameters
relevant to SV analysis (see Note 4). The program SEDFIT
created by P. Schuck and available free (http://sedfitsedphat.
nibib.nih.gov), for the c(s) analysis of SV experiments [14]. The
program SEDPHAT created by P. Schuck and available free
(http://sedfitsedphat.nibib.nih.gov), for the global analysis of
different AUC [21] and other biophysical experiments
[22, 23], for the analysis of isotherms [17] and to generate
simulated data sets. The program GUSSI created by
C. Brautigam and available free (http://biophysics.swmed.
edu/MBR/software.html), for calculations (e.g., the integration of the c(s) peaks, the output of isotherm data files), and
publication quality illustrations of AUC experiments [24].
3 Methods
3.1 Data Acquisition
1. Prepare the analytical ultracentrifugation cells assemblies. Fill
the sample compartments with 400 μL (12 mm path length
centerpiece) or 100 μL (3 mm path length centerpiece) of
protein solutions and the solvent compartments with 420 or
Heterogeneity and Affinity Interaction Analysis by Sedimentation Velocity
161
2.1 Protein Samples
1. BSA (SIGMA) was dissolved at 5 mg/mL in 20 mM Na-K
Phos 150 mM NaCl pH 7 and filtered at 45 μm. BSA1 sample
was obtained by five times dilution to 1 mg/mL. BSA2 sample
at 0.8 mg/mL was obtained from the main elution peak of
500 μL BSA stock sample injected at 0.5 mL/min on a Superose 12 10/300 GL (GE Healthcare) size exclusion chromatography column.
2. Recombinant BTG2 fused to glutathione S transferase
(GST-BTG2) and PAPB proteins were purified by affinity and
size exclusion chromatography [20]. Stocks of GST-BTG2
protein at 1.1 mg/mL and PABP at 5.1 mg/mL in buffer
10 mM K phosphate, pH 7.5, 100 mM NaCl, 0.5 mM TCEP
were used in this study (see Note 3). In the dilution series,
mixtures contain a fixed concentration of GST-BTG2 (5 μM)
and a range of seven PABP concentrations to give molar ratio
(PABP/GST-BTG2) of 1:2, 1:1, 2:1, 4:1, 8:1, 16:1, and 32:1.
2.2 Instrument
and Sample Cells
1. An analytical ultracentrifuge (Optima XLI Beckman) and rotor
(8-hole AnTi-50, Beckman). Analytical ultracentrifugation cell
assemblies equipped with sapphire windows and two-channel
Titanium, Aluminum or Epon centerpieces of 1.5, 3, and/or
12 mm optical path length (Nanolytics or Beckman).
2. Programs: The program SEDNTERP created by D. Hayes,
T. Laue, J. Philo and available free (http://bitcwiki.sr.unh.
edu/index.php/Downloads) for calculating the parameters
relevant to SV analysis (see Note 4). The program SEDFIT
created by P. Schuck and available free (http://sedfitsedphat.
nibib.nih.gov), for the c(s) analysis of SV experiments [14]. The
program SEDPHAT created by P. Schuck and available free
(http://sedfitsedphat.nibib.nih.gov), for the global analysis of
different AUC [21] and other biophysical experiments
[22, 23], for the analysis of isotherms [17] and to generate
simulated data sets. The program GUSSI created by
C. Brautigam and available free (http://biophysics.swmed.
edu/MBR/software.html), for calculations (e.g., the integration of the c(s) peaks, the output of isotherm data files), and
publication quality illustrations of AUC experiments [24].
3 Methods
3.1 Data Acquisition
1. Prepare the analytical ultracentrifugation cells assemblies. Fill
the sample compartments with 400 μL (12 mm path length
centerpiece) or 100 μL (3 mm path length centerpiece) of
protein solutions and the solvent compartments with 420 or
Heterogeneity and Affinity Interaction Analysis by Sedimentation Velocity
161