B-Per reagent: from Pierce, Cat
This procedure comes to replace sonication of the bacteria. It is often used for initial screening of expression conditions and not for large scale preparation of protein. B-Per doesn’t properly dissolves aggregates, so finding your protein in the pellet doesn’t necessarily mean the protein is in inclusion bodies. Pierce has special kits for His-tag and GST isolations from B-Per, since most home-made procedures for binding and elution for GST or His tag do not work well with this reagent.
1. Pellet bacterial cells by centrifugation at 5000 RPM for 10 minutes in a microcentrifuge.(The cells can either be used fresh or frozen at -70oc .)
2. Resuspend the cells in 300cl of B-PER reagent by either vigorously vortexing the mixture or by pipetting up and down until the cell suspension is homogenous. Vortex for 1 more minute. (If sup is too viscous add 3U of DNase in final concentration of 10mM MgCl, 10min in 37ºC or 30min 4ºC)
3. Centrifuge at 13000 RPM for 10min, 4ºC to separate the soluble proteins from the insoluble proteins.
4. Collect the supernatant (soluble fraction ) and resuspend
the pellet (insoluble fraction)in 300ml of PBS.
Use 10ml each of the soluble and insoluble fraction
for SDS-PAGE or western blotting assay to determine the solubility
of the recombinant protein. If purification of inclusion bodies is required,
proceed to step 5.
When loading on gel: Add 100ml of 4Xsample buffer (+100mM DTT), boil 95-100ºC for 5min, and then load on gel.
5. For inclusion body purification , add lysosym (6ml of 10 mg/ml stock solution) to the resuspend pellet(insoluble fraction generated in step 4 ) to a final concentration of 200mg/ml, and vortex for 1 minute. Add 1 ml of 1:10 diluted B-PER reagent to the suspension and vortex for 1 minute.
6. Collect inclusion bodies by centrifugation at 13000 RPM for 10 minutes. Resuspend the pellet in 1 ml of 1:10 diluted B-PER reagent and vortex for 1 minute.
7. Repeat step 6 two more times.
8. Resuspend the final inclusion body pellet in 300 ml
of ddH2O or desired buffer. Use 10-20 ml
of sample for SDS-PAGE assay to determine the purity of the inclusion body