Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
Separating Soluble from Insoluble Induced Cell Fractions and
Preparing SDS-PAGE Samples
Student Workstations
Each student team requires the following items to separate soluble from insoluble lysate fractions and
prepare soluble and insoluble fraction samples for SDS-PAGE analysis:
Material Needed for Each Workstation Quantity
Thawed cell lysate 1 ml
Lysis buffer 2 1 ml
Screwcap microcentrifuge tube, 1.5 ml 2
Microcentrifuge tubes, 2 ml 3
Laemmli sample buffer (left over from the previous activity) 1 ml
20–200 µl adjustable-volume micropipet and tips 1
100–1,000 µl adjustable-volume micropipet and tips 1
10 ml syringe 1
22 gauge syringe needle 1
Marking pen 1
Common Workstation Quantity
Water bath or dry bath set to 95°C 1
Microcentrifuge with variable speed setting >16,000 x g 1
Separate soluble from insoluble cell lysate components
1. Make sure that your lysate is balanced with either another student’s lysate fraction or with a
microcentrifuge tube filled with water.
2.
Separate the insoluble fraction of the lysed cells from the soluble fraction by centrifugation at
16,000 x g for 20 minutes.
3. Label a clean 2 ml microcentrifuge tube “ Soluble fraction” with your initials. Gently pour the
supernatant from the “Lysed Cells” tube into the “Soluble fraction” tube being very careful not to
decant any of the insoluble fraction (the opaque, viscous blob that is mainly composed of genomic
DNA) into your soluble fraction.
Note: The insoluble fraction does not necessarily adhere to the tube so extreme care is needed when
decanting.
4. Relabel the “ Lysed Cells” microcentrifuge tube containing the remaining insoluble fraction “Insoluble
fraction.”
5. Add 1 ml of Lysis buffer 2 to the “Insoluble fraction” tube and resuspend the pellet by shearing with
a syringe needle.
Note: The insoluble fraction contains a large quantity of genomic DNA that can be quite viscous,
making resuspension difficult. Using a 22 gauge needle, the insoluble fraction can be pulled up into a
3 ml syringe and expelled from the syringe into a clean tube multiple times to decrease the viscosity of
the DNA. Ensure that the needle is disposed of properly in a sharps container.
Remove 50 µl of “
6. Soluble fraction” and place it in a clean 1.5 ml screwcap microcentrifuge tube
labeled “Soluble PAGE” with your initials. Add 50 µl Laemmli sample buffer and mix thoroughly.
Chapter 4: 11 ml Culture Protocol for
Centrifugation Purification
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CHAPTER 4
11 ml CULTURE
PROTOCOL