- Visualize the TLC plate by transferring plate to a new TLC
tank containing iodine pellets (or by preferred visualization
technique). - Mark the location of the cholesterol standard, and using a
straight razor blade, scrape the silica onto a piece of weigh
paper before transferring to a 7 mL scintillation vial. - Add 4–5 mL of scintillation fluid and count using a liquid
scintillation counter (LSC). - Perform a bicinchoninic acid assay (BCA) with a protein deter-
mination kit (or equivalent protein quantification assay) of
original cell suspension in1PBS. - For BCA assay: prepare BSA (bovine serum albumin) protein
serial dilutions ranging from 0 to 2 mg protein/mL for known
protein standards. - Pipette 25μL of each protein standard into a 96-well plate to
generate a standard protein concentration curve (known pro-
tein value absorption vs. known concentration). - Pipette 25μLof1PBS into the 96-well plate as a blank.
- Pipette 25μL of each experimental sample into the 96-well
plate. - For each reaction, add 196μL of reagent A to 4μL of reagent B
(20:1 ratio); scale this master mix to accommodate all standard,
blank, and sample wells (in duplicate if preferable). - Add 200μL of 20:1 working reagent (AþB) to each well.
- Incubate at 37C for 30 min.
- Read each samples absorbance at 562 nm on a microplate
reader. - Generate a line of best fit for the absorbance value versus
known protein concentrations for all the protein standards:
y¼xþby¼absorbance.
m¼slope of line.
x¼protein concentration.
b¼yintercept where the line crosses theyaxis.- Generate equation solving for “x”:
x¼ðÞyb=m- Subtract the averaged absorbance value for the duplicates of 1
PBS samples from each experimental sample replicate.
486 Nicholas D. LeBlond and Morgan D. Fullerton