(1) Clarify the protein solution (in most cases the lysates) by centrifugation.
(2) Transfer the supernatant into an ice cold beaker with a magnetic bead.
(3) Note the exact amount of the supernatant.
(4) Keep the beaker chilled by placing it in an ice tray.
(5) Transfer the beaker with the ice tray onto a magnetic stirrer
(6) Weigh the amount of ammonium sulfate to be added. The amount depends on the volume of the solution and the percentage saturation of the salt needed. Refer to the precipitation chart. In case of protein purification, a step precipitation is carried out.
(7) Slowly add the ammonium sulphate with stirring. One needs to be careful as the addition of the salt should be very slow. Add a small amount at a time and then allow it to dissolve before further addition.
(8) Keep it on the stirrer for 1hr precipitation to occur in ice.
(9) Centrifuge at 10,000g for 15min at 4oC.
(10) The pellet contains the precipitated protein which could be dissolved in a suitable buffer for further analysis and purification.
(11) For a second round of precipitation of a different protein, the supernatant is again used and the above same steps are followed.
(2) Transfer the supernatant into an ice cold beaker with a magnetic bead.
(3) Note the exact amount of the supernatant.
(4) Keep the beaker chilled by placing it in an ice tray.
(5) Transfer the beaker with the ice tray onto a magnetic stirrer
(6) Weigh the amount of ammonium sulfate to be added. The amount depends on the volume of the solution and the percentage saturation of the salt needed. Refer to the precipitation chart. In case of protein purification, a step precipitation is carried out.
(7) Slowly add the ammonium sulphate with stirring. One needs to be careful as the addition of the salt should be very slow. Add a small amount at a time and then allow it to dissolve before further addition.
(8) Keep it on the stirrer for 1hr precipitation to occur in ice.
(9) Centrifuge at 10,000g for 15min at 4oC.
(10) The pellet contains the precipitated protein which could be dissolved in a suitable buffer for further analysis and purification.
(11) For a second round of precipitation of a different protein, the supernatant is again used and the above same steps are followed.
No comments:
Post a Comment