Lysates:
- Non-transfected HEK cells
- FLOT1/2 transfected HEK cells
- Rat brain
- Rat cerebellum
- Swine optic nerve
All of them were lysed in RIPA buffer + protease inhibitors (30 min on ice + centrifuge 15 min at 4ºC)
Procedure for protein quantification:
- Dilute the lysates 1/10 with RIPA buffer
- Add 10 microl of the diluted lysate in a 96 well plate (duplicate all the lysates: 2 wells/lysate). Blank: 2 wells with 10 microl of RIPA
- Add 290 microl of cytoskeleton buffer in all the wells used
- Incubate for 1 min at RT
- Read the plate at 595 nm
To calculate the amount of protein:
- Calculate the mean of the 2 absorbances of each sample. Substract the mean absorbance of the blank (RIPA alone)
- Multiply the result by 3’75
- Multiply the result by 10 (dilution factor) –> the result wil be in mg/ml
RESULTS
- Non-transfected HEK cells: 11,75 mg/ml
- FLOT1/2 transfected HEK cells: 3,03 mg/ml
- Rat brain: 17,26 mg/ml
- Rat cerebellum: 8,23 mg/ml
- Swine optic nerve: 0,97 mg/ml
