Allis Lab:Notebook/SURF/2010/06/11

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DETECTING PROTEIN EXPRESSION OF INTRODUCED PLASMID:

OBJECTIVE: to determine if there are enough proteins to pull down GENERAL APPROACH: grow culture and induce protein expression (1) then run in SDS-PAGE (2) DETAILS: 1) transformed E. coli (method?) with "Rosetta" (a proprietary mammalian tRNA containing bacteria to facilitate for plasmid translation). Can be grown from 20-37*C with varying incubation, however higher temperatures produces inclusion bodies (i.e. aggregates of proteins) that are insoluble thus inactive (?). Optimal yield can be obtained at 25*C, 6 hrs (Simon E.). Alternative method: Can induce autoinduction (see autoinduction protocol). 2) SDS-PAGE buffers was made according to Qiagen's handbook (see SURF folder) and BR's recipe (JHU).Samples were prepared according to SE's (RU) method: whole cell lysate protocol soluble protein protocol -spin 1 ml (preinduction) / 500 uL (post induction) of bacterial broth for 1 min at 10k rpm/~10k g using a table top centrifuge. -add 100 uL 1x SDS sample/loading buffer. -vortex/resuspend (preferred). -boil 5' to lyse cells. -sonicate in waterbath for 10'. -boil 5' -spin 5' @ 13k rpm (max). -load 10 uL on gel (15/12% gels). -spin 500 uL of culture, discard supernatant. -add 100 uL lysis buffer (50mM MOPS phi, 250mM NaCl, 1% Tritonx-100,1mM PMSF note: add PMSF fresh due to half-life). -sonicate 10' with ice water (to decrease protease activity). -spin 5' at max @ 4*C. -transfer 50 uL supernatant to new tube. -ass 10 uL 6x SDS loading buffer. -boil 5'. -run 10-20 uL on gel.

note: Samples will not appear identical while running due to differences in buffers.