Team:WashU/Protocols/Celllysis

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<!--Thanks to UCSD for the above instructions.  The original source can be found here: http://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/lysis_buffer_additives/ under the "General lysis buffer" section. EDTA was substituted for DDT and NaCl was not included as we did not have it currently available at the time of creation-->
<!--Thanks to UCSD for the above instructions.  The original source can be found here: http://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/lysis_buffer_additives/ under the "General lysis buffer" section. EDTA was substituted for DDT and NaCl was not included as we did not have it currently available at the time of creation-->
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==Cell Lysis and Protein Purification Prep==
==Cell Lysis and Protein Purification Prep==

Revision as of 19:36, 2 August 2012


Cell Lysis and Protein Purification Prep

Buffer:

  1. The appropriate amount of tris(hydroxymethyl)aminomethane (Tris) was weighed to create a 50 mM solution. (6.057g/ liter of solution).
  2. Similarly, ethylenediaminetetraacetic acid (EDTA) was weighed out to create a 1 mM solution. (0.292g/ liter of solution).
  3. The two substances (Tris and EDTA) were dissolved in deionized water. (The solution had some difficulty dissolving and so was placed in a 37°C water bath for a few hours.)
  4. Now, the solution was pH-ed using 1 M HCl to a pH of 7.5. (NaOH may be added in the event of an overshoot.)


Lysis:

  1. Spin down the cells desired in a microcentrifuge for 1 minute at 12,000RPM. Discard supernatant and resuspend the culture in the buffer above.
  2. Sonicate or use some other method to extract proteins.
  3. Refer back to the SDS-PAGE protocol to see how to load the gel.


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