Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
9.
10.
Carefully apply another 75 µl of each of your eluted fractions chosen in step 1 to the correspondingly
labeled columns and 2 ml microcentrifuge tubes and centrifuge for four minutes at 1,000 x g to
produce a final volume of 150 µl of desalted GST-DHFR-His for each elution fraction you chose.
Discard the columns.
Preparing SDS-PAGE samples of desalted GST-DHFR-His fractions
1.
2.
3.
In a clean 1.5 ml screwcap microcentrifuge tube, combine 25 µl of Laemmli sample buffer with 25 µl of
your desalted GST-DHFR-His fraction A and label the tube “Desalted A PAGE” with your initials.
In a clean 1.5 ml screwcap microcentrifuge tube, combine 25 µl of Laemmli sample buffer with 25 µl of
your desalted GST-DHFR-His fraction B and label the tube “Desalted B PAGE” with your initials.
In a clean 1.5 ml screwcap microcentrifuge tube, combine 25 µl of Laemmli sample buffer with 25 µl of
your Desalted GST-DHFR-His fraction C and label the tube “Desalted C PAGE with your initials.
4. Heat the three SDS-PAGE samples at 95°C
for five minutes.
5. Store the SDS-PAGE samples at –20°C until ready to analyze via SDS-PAGE.
Cover with parafilm and store the
6. Desalted A, B and C fractions at 4°C until ready to analyze via
spectroscopy, SDS-PAGE, and enzymatic activity assay. Do not freeze your fractions.
Chapter 7: Purification Protocol for BioLogic LP System
155
CHAPTER 7
BIOLOGIC LP SYSTEM
PROTOCOL