Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
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 into a 10
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.
7. Remove 50 µl of “ 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.
8. Remove 50 µl of “ Insoluble fraction” and place it in a clean 1.5 ml screwcap microcentrifuge
tube labeled “Insoluble PAGE” and your team’s initials. Add 50 µl Laemmli sample buffer and mix
thoroughly.
9. Heat the “ Soluble PAGE” and “Insoluble PAGE” samples at 95°C for five minutes.
Note: Make sure that you are heating your blue SDS-PAGE samples and not your actual soluble and
insoluble fractions!
10. Store the SDS-PAGE samples “ Soluble PAGE” and “Insoluble PAGE” at –20°C until ready to
analyze via SDS-PAGE.
11. Store the soluble fraction and insoluble fractions at –20°C until ready to purify the soluble fraction.
Chapter 5: 100 ml Culture Protocol for
Chromatography Instrumentation Purification
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CHAPTER 5
100 ml CULTURE
PROTOCOL