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 volume to new tube being careful not to disturb the pellet at the bottom of each tube.


Plate reader measurements

RFP signal

  1. BL09 1 = 109
  2. BL09 2 = 610
  3. BL09 negative ctrl (no DNA) = 47


Bradford assay

  • Used 5 μL for each BL09 sample
  • OD 590 readings:
  1. Standard 1 (0 μg) = 0.25
  2. Standard 2 (1 μg) = 0.266
  3. Standard 3 (0 μg) = 0.295
  4. Standard 4 (0 μg) = 0.356
  5. Standard 5 (0 μg) = 0.432
  6. Standard 6 (0 μg) = 0.608
  7. BL09 1 = 0.486
  8. BL09 2 = 0.481
  9. 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




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