- 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
- BL09 1 = 109
- BL09 2 = 610
- BL09 negative ctrl (no DNA) = 47
- 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
||Raw Fluor. (100 uL)
||Fluor/ μg protein
| BL09 1
| BL09 2
| BL09 negative