Haynes Lab:Notebook/Engineering PC-TFs/2014/09/15: Difference between revisions
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==Summary== | ==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 | |||
[[Image:CG091514_Bradford.jpg|400px]] | |||
Final results: | |||
{| {{table}} | |||
| 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 | |||
|} | |||
Latest revision as of 00:17, 27 September 2017
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Summary
Protein extraction from transfected cells
Plate reader measurements RFP signal
Standard curve
|