Freeze-Thaw Cell Lysis Protocol for Protein Samples

Reagents and Materials

• IPTG (Isopropyl-Beta-D-Thiogalactoside)
• Lysis Buffer (50mM NaH2PO4 + 500mM NaCl)/li>
• PMSF (phenylmethylsulfonyl fluoride)
• Lysozyme
• DTT (dithiothreitol)
• Falcon tubes
• Dry ice

Protocol

1. Centrifuge cells at max for 10 minutes. Discard supernatant
2. Add 250 µL Lysis Buffer, 2.5 µL PMSF, 5 µL Lysozyme, and 1 µL DDT. Re-suspend pellet
3. Leave on ice for 30 min
4. Put samples in dry ice for ~ 3 minutes, and then transfer to 37°C water bath for ~3 minutes. Repeat this cycle 6 times.
5. Centrifuge at max for 10 minutes
6. Transfer supernatant to a 1.5 mL microcentrifuge tube. Re-suspend the pellet in 250 µL of Lysis Buffer and transfer to a separate microcentrifuge tube
7. Run a Bradford Assay on the samples if the protein amount in the gel must be constant
8. Add appropriate amount of dye and sample (40 µL total). If Bradford was not performed, use 30 µL of protein sample and 10 µL of 4x dye
9. Boil the sample/dye mixture for 5 minutes at 95°C
10. Load 30 - 40 µL of sample into gel. Run gel at 100V until the sample moves out of the wells, then run at 150V