Protein Expression and Purification Series - Bio-Rad

CHAPTER 3A
ADVANCE PREP
CENTRIFUGATION PROCES
Protein Expression and Purification Series
Advance Preparation for SDS-PAGE Electrophoresis Activities
In this SDS-PAGE Electrophoresis Laboratory, the uninduced cells, induced cells, soluble fraction of the
induced cell lysate, insoluble fraction of the induced cell lysate, flowthrough, wash, eluate and desalted
eluate fractions from the chromatographic separations will be separated and visualized using SDS-PAGE.
SDS-PAGE Electrophoresis Laboratory Checklist
Components from Protein Expression
and Purification Series Where Provided (✔)
10x Tris/Glycine/SDS buffer (TGS) SDS-PAGE Electrophoresis Module ❒
Precision Plus Protein Dual Color standards SDS-PAGE Electrophoresis Module ❒
Bio-Safe Coomassie stain SDS-PAGE Electrophoresis Module ❒
Required Accessories (Not Provided) Quantity (✔)
Microcentrifuge with variable speed setting >16,000 x g 1 ❒
2–20 µl adjustable-volume micropipets and tips 12 ❒
20–200 µl adjustable-volume micropipets and tips 12 ❒
Water bath or dry bath set to 95°C 1 ❒
Marking pens 12 ❒
Vertical electrophoresis chambers and power supplies 3–12 ❒
4–20% Mini-PROTEAN ® TGX precast gel 12 ❒
Gel staining trays 12 ❒
Tasks to Perform Prior to the Lab
1. Tris/Glycine/SDS (TGS) running buffer: One Mini-PROTEAN Tetra cell with two gels requires 700
ml of 1x TGS running buffer. One Mini-PROTEAN Tetra cell using the companion running module to
run four gels requires 1.1 L of 1x TGS running buffer. To make 10L of 1x TGS running buffer, mix 1 L of
10x TGS with 9 L of distilled water. Store at room temperature.
Please refer to Appendix E for more details on using the Mini-PROTEAN Tetra cell. If using other
vertical gel boxes please refer to the instruction manual for that instrument for details on setting up the
apparatus.
Tip: You may want to prepare 1–2 L of extra 1x TGS buffer in case your gel boxes leak after assembly.
If you do have a leak, the outer chamber of the gel box can be filled to just above the inner small
plates to equalize the buffer levels in both reservoirs. This requires approximately 1,200 ml of 1x TGS
buffer per gel box and is a more convenient fix than reassembling the apparatus mid-lesson.
2. Aliquot 15 µl of Precision Plus Protein Dual Color standards into a 2 ml microcentrifuge tube for each
workstation.
3. Set up student workstations according to the student workstation list on page 69.
68 Chapter 3A: Advance Preparation
for Centrifugation Protocols