Haynes Lab:Notebook/Engineering PC-TFs/2014/09/15

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Summary Protein extraction Plate reader measurements: RFP and Bradford (protein) Protein extraction from transfected cells Warmed media, PBS, Trypsin to 37°C. Warmed protease inhibitor cocktail (protease arrest) and protein extraction buffer to room temperature. Removed six well plate from incubator. Two wells (W1, W2) have transfected cells. Aspirated off media from each well. Washed first three wells with 1mL PBS (W1, W2, Negative control). Aspirated off PBS. Added 0.5mL Trypsin. Incubated for 3 minutes at 37°C. Added 5mL media to each well. Pipetted up and down to remove remaining adherent cells. Transferred cells from each well into their own 14mL conical tube. Pelleted cells by spinning at 1000 x g for 3 minutes. Aspirated off supernatent leaving ~100μL covering the pellet. Resuspended cells in tube by flicking. Placed these cells on ice when not in direct use for following steps. Labeled three 1.5mL tubes (W1, W2, Neg). Added 500μL protein extraction buffer to each tube. Added 5μL protease inhibitor cocktail (100x) to each tube. Inverted to mix. Aspirated 505μL combination mixture and dispensed into corresponding tube containing cell pellet. Pipetted up and down to mix. Aspirated total buffer, protease inhibitor, resuspended cell pellet and dispensed back into corresponding 1.5mL tube. Repeated for other two samples. Centrifuged samples at max rpm for five minutes in cold room. Labeled three new 1.5mL tubes. Transferred supernatant to new tube being careful not to disturb the pellet at the bottom of each tube. Used in Bradford Assay and stored in the -20°C. Plate reader measurements RFP signal Followed the fluorescent protein assay: http://openwetware.org/wiki/Haynes:ExtFluorProtein Used 100 μL per well Results: BL09 1 = 109 BL09 2 = 610 BL09 negative ctrl (no DNA) = 47 Bradford assay Followed Bradford Assay protocol: http://openwetware.org/wiki/Haynes:Bradford Used 5 μL for each BL09 sample OD 590 readings: Standard 1 (0 μg) = 0.25 Standard 2 (1 μg) = 0.266 Standard 3 (0 μg) = 0.295 Standard 4 (0 μg) = 0.356 Standard 5 (0 μg) = 0.432 Standard 6 (0 μg) = 0.608 BL09 1 = 0.486 BL09 2 = 0.481 BL09 negative = 0.471 Standard curve Final results: Sample Protein μg/μL Protein*100 μL Raw Fluor. (100 uL) Fluor/ μg protein BL09 1 10.42 1041.96 109 0.104610111 BL09 2 10.20 1019.64 610 0.598248687 BL09 negative 9.75 975.00 47 0.048205128