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Cell lysis

BC Byoungsan Choi

Last updated date: May 8, 2020 Views: 2137 Forks: 0

original An abbreviated version of this protocol was published in eLife in Apr, 2020
Single-molecule functional anatomy of endogenous HER2-HER3 heterodimers
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  1. Prepare lysis buffer base: 50mM HEPES-NaOH [pH 7.4], 150mM NaCl, 10% glycerol (v/v), 2% protease inhibitor cocktail (v/v) (SigmaAldrich P8340), 2% tyrosine phosphatase inhibitor cocktail (v/v; to preserve endogenous phosphorylation level) (SigmaAldrich P5726)

  2. Dissolve mild detergent (digitonin or GDN at 1% w/v) into the lysis buffer base.

  3. Add complete lysis buffer to the cell pellet (We used ~400ul of complete lysis buffer to lyse 10^7 of SKBR3 cell).

  4. Using micro-pipet, resuspend cell pellet completely.

  5. Incubate for 10 min at 4℃.

  6. Repeat step 4~5, 3 times.

  7. Centrifuge the mixture at 15000g, 4℃, for 10 min.

  8. Collect supernatant.

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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Choi, B(2020). Cell lysis. Bio-protocol Preprint. bio-protocol.org/prep306.
  2. Choi, B., Cha, M., Eun, G. S., Lee, D. H., Lee, S., Ehsan, M., Chae, P. S., Heo, W. D., Park, Y. and Yoon, T.(2020). Single-molecule functional anatomy of endogenous HER2-HER3 heterodimers. eLife. DOI: 10.7554/eLife.53934

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