Team:Exeter/lab book/proto/13

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Latest revision as of 23:51, 26 September 2012

Protocol 13

SDS-PAGE Protocol

SDS-PAGE was run using 10% NuPAGE® Novel® Bis-Tris Mini Gels.

  • Prepare a master-mix containing (procedure for 1 sample only):
    • 2x NuPAGE® LDS Sample Buffer (4x provided)
    • 2x Sample Reducing Buffer (10x provided)
    • Top up to 50µL using MilliQ H2O

  • Heat samples at 70oC for 10 minutes.

  • Prepare 1 x SDS Running Buffer by adding 50mL 20 x NuPAGE® MES or MOPS SDS Running Buffer to 950mL of deionised water.

  • Load the appropriate concentration of your protein sample on the gel.

  • To load the buffer, fill the Upper (inner) Buffer Chamber with 200mL 1 x NuPAGE® SDS Running Buffer. Fill the Lower (outer) Buffer Chamber with 600mL 1 x NuPAGE® SDS Running Buffer.

  • Then run the SDS-PAGE gel at 200V constant for 35 minutes (for MES Buffer) or 50 minutes (for MOPS Buffer).

  • Once finished, stain using SimplyBlue™ SafeStain, leave for approximately an hour, and then wash using MilliQ H2O for direct visualisation.

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