Lysate preparation for western blot

Last edited Tue 22 Aug 2023

Recipes for buffers referenced here are as follows:

• 1% SDS hot lysate buffer

  • 10 mM Tris-HCl (ph 8.0)
  • 1% SDS
  • 1.0 mM Na-Orthovanadate
  • ddH₂O

• 2x Sample buffer

  • 62.5 mM Tris-HCl (pH 6.8)
  • 2% SDS
  • 0.01% Bromophenol Blue
  • 25% Glycerol
  • 710 mM ß-Mercaptoethanol
  • ddH₂O

Stage 1 - 1% SDS hot lysate buffer preparation

Steps

Discard the medium in the flask and wash once with pre-cold PBS.

Add 3 mL pre-cold PBS per flask and collect cells with cell scraper.

Add 12 mL pre-cold PBS to make sure all the cells detach from the flask.

Transfer collected cells to 50 mL centrifuge tubes, centrifuge at 300 x g for 5 min.

Discard supernatant, wash twice with pre-cold PBS.

Heat 1%SDS hot lysis buffer to 90-95℃. Re-suspend the cells with the buffer.

Pipette the cells in boiling buffer for 1 minute. Then boil them at 90-95℃ for 10-20 min.
(Mix the samples periodically during the boiling)

Use an ultrasonic cell disruptor to break all cell clusters until the lysate becomes clear.

• Ultrasound time 3 s, 10 s interval, ultrasonic 5-15 times, ultrasonic power: 40 kW

Centrifuge for 5-10 min at 15000-17000 x g and discard cell pellet.

Steps

Shatter the frozen tissue with pre-cold scissors.

• Grind tissue into powder with a pre-cold mortar.

Heat 1% SDS hot lysis until bubbling.

Add 1% SDS hot cell lysis according to the tissue amount to resuspend cells.

• Add by pipetting in boiling water for 10-20 min.

Use ultrasonic cell disruptor to break all cell clusters until the lysate becomes clear.

• Ultrasound time 3 s, 10 s interval, ultrasonic 5-15 times, ultrasonic power: 40 kW

Centrifuge for 5-10 min at 15000-17000 x g and discard cell pellet.

Stage 2 - RIPA lysate preparation

Steps

Discard the medium in the flask and wash once with pre-cold PBS.

Add 3 mL pre-cold PBS per flask and collect cells with cell scraper.

Add 12 mL pre-cold PBS to make sure all the cells detach from the flask.

Transfer collected cells to 50 ml centrifuge tubes, centrifuge at 300 x g for 5-10 minutes.

Discard supernatant, wash twice with pre-cold PBS.

Add RIPA buffer according to the cell amount to resuspend cells.

• Place on ice for 15 min.

Use ultrasonic cell disruptor to break all cell clusters until the lysate becomes clear.

• Ultrasound time 3 s, 10 s interval, ultrasonic 5-15 times, ultrasonic power: 40 kW

Centrifuge for 5-10 min at 15000-17000 x g and discard cell pellet.

Steps

Shatter the frozen tissue with pre-cold scissors.

• Grind tissue into powder with a tissue grinding instrument

Add RIPA buffer according to the tissue amount to re-suspend tissue.

• Place on ice for 15 min

Use ultrasonic cell disruptor to break all cell clusters until the lysate becomes clear.

• Ultrasound time 3 s, 10 s interval, ultrasonic 5-15 times, ultrasonic power: 40 kW

Centrifuge for 5-10 min at 300 x g and discard tissue pellet.