Dialyzed to obtain soluble refolding σ32 Monomer experiments
Dialyzed to obtain soluble refolding σ32 Monomer experiments
This experiment describes what dialysis conditions lead to the highest yield of soluble refolded Σ32 monomer. This experiment is from the guide to protein purification and characterization by Zhu Houzhu.
Operation method
Dialysis of soluble refolded σ32 monomer experiments
Materials and Instruments
Tris NaCl Move Materials and equipment For more product details, please visit Aladdin Scientific website.
CentriconTM-10 Centrifugal Concentrator High-efficiency Liquid Chromatography Columns with Large or Small Displacement
CentriconTM-10 Centrifugal Concentrator (Amicon, Inc.)
High performance liquid chromatography column TSK-GEL G3000 PWXL (7.8x300 mm) (TosoHaas)
50 mmol/L Tris(pH7.9)+200 mmol/L NaCl
Operating Procedures
Detection of soluble monomer formation by size exclusion HPLC chromatography
1) Dialyze the sample under the conditions of the proposed test.
2) Remove the sample from the dialysis bag and centrifuge at 13000r/min for 10 min to clarify. Retain the supernatant.
3) If the protein concentration of the sample is too low at the time of dialysis, it can be concentrated in a Cenricon-10 centrifuge. Add 2.0 ml of dialysis sample to a Centricon-10 tube and centrifuge at 13000 r/min for 90 min, or until the volume is reduced to 1/5 to 1/10 of the original volume.
Note: It is necessary to select Centricon tubes of a certain pore size so that water and salt can pass through the membrane under centrifugal force and retain σ32. Centricon-30 and Centrioon-100 tubes have larger pore sizes and are faster to concentrate, but Centricon-10 tubes were used in the present experiment with a nominal MWCO of 10,000 Da because the molecular weight of σ32 is 32,000 MWCO and the molecular weight of σ32 is 32,000 MWCO. The Centricon-10 tubes were used in this experiment, which have a nominal MWCO of 10,000 Da, because the molecular weight of σ32 is 32,000 Da and some of the σ32 will pass through the larger pores. The tubes can also be used to change the salt concentration of a sample by simply concentrating the sample by a factor of 10 and bringing it back to the original volume with another buffer. Concentrators capable of handling smaller (0.5 ml; Micrecon model) and larger (20 ml; Centriprep model) volumes are also available from Amicon.
4) Feed 100-400ul of supernatant to a size exclusion column, TSK-GELG3000PWXL (7.8x300 mm), which has been previously equilibrated with 50 mmol/L Tris (pH 7.9) + 200 mmol/L NaCl at a flow rate of 1 ml/min and eluted under the same conditions, and collect the 0.5-ml component in 30 min. min.
Note: Salt is added to prevent weak ionic binding of proteins to the polymer matrix, which can distort elution times and prevent size exclusion chromatography from working properly.
5) Detect the effluent from the column at 280mn with a UV detector to estimate the proportion of material that will effuse as soluble monomers.
NOTE: The HPIX size exclusion column should be calibrated by passing a set of proteins of known molecular weight through the column under the same elution conditions. The molecular weight of the protein eluted in the analysis of the σ32 dialysis sample can be found by inserting the logarithm of the molecular weight of the molecular weight standard reference plotted against its peak time.
6) If necessary, analyze selected fractions by SDS-PAGE electrophoresis to confirm the consistency of elution peaks.