Protocol

Abbreviation

UTSW_Purification_HPLC

Name

using FPLC

Laboratory name

University of Texas Southwestern Medical Center

Type

purification

Description

FPLC Protocol

Autoclave LB medium and 50ml flasks
Inoculate glycerol stock in 50ml of LB medium + Antibiotic O/N @ 37°C
Amp – 50µl of 100mg/ml [final conc. 200µg/ml]
Kan - 50µl of 100µg/ml [stock – 50mg/ml]
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Prepare 1X TB broth 1L in 2 flasks [10 pouches in 5L water] Autoclave flasks.
Centrifuge culture 4000rpm, 40mins @ 4°C. Resuspend pellet in 5ml of ddH2O
Inoculate cell culture + Antibiotics Amp – 2ml of 100mg/ml [final conc. 200µg/ml]
Kan - 2ml of 100µg/ml [stock – 50mg/ml] in TB broth until OD600nm is ~ 0.6-0.8
Decrease temp to 20ºC and induce protein expression with 1ml of 1M IPTG (final concentration 1mM)
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Weight empty centrifuge bottles
Centrifuge @ 4°C, 4000rpm, and 40mins.
Discard supernatant, invert tubes on paper towel for a min.
Weight the pellets n store in -80 °C
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Centrifuge bottles after taking from -80 °C rinse under DI water and Lyses buffer + Protease inhibitor and vortex + Mix with syringe and finally use cell disrupter and lyse cells 5 times and collect samples in centrifuge tubes containing brown ring in caps.
Lysis buffer – 20mM Hepes + 100mM NaCl + 0.03% Brj-35 + 5mM β-mercaptoethanol
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Centrifuge @ 10,000 rpm, 40mins, and 4°C
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Collect samples (pellet facing upside) and Filter using 0.8µ
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Run Ni-column, Run SDS-PAGE gel, Pool fractions
Ni Buffer A (1L) - 20mM Hepes pH 8 + 500mM NaCl
Ni Buffer B (1L) - 20mM Hepes pH 8 + 500mM NaCl + 500mM Imidazole
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Cleave His-Tag using TEV Protease, Dialysis O/N
Dialysis Buffer – 500mM NaCl + 5% Glycerol + 10mM Hepes + 1mM DTT
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Run Q-column, Run SDS-PAGE gel, Pool fractions
Q Buffer A (1L) - 20mM Hepes pH 8 + 1mM DTT
Q Buffer B (500ml) - 20mM Hepes pH 8 + 1mM DTT + 1M NaCl
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Concentrate Protein to 5ml
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Run Gel-filtration column, Run SDS-PAGE gel, Pool fractions
Gel filtration Buffer (1L) - 200mM NaCl + 50mM Hepes + 1mM DTT
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Concentrate ~ 20mg/ml, freeze protein