The colorimetric method can determine the amount of total protein at any point in time. Continuous observation over a period of time can be used to determine net protein accumulation or loss (i.e., synthesized protein-degraded protein). Protein synthesis at this time can be measured in liquid flash by incubating cells with radiolabeled amino acids, such as 3H-leucine or 35S-methionine, and measuring the radioactivity per gram of protein incorporated into 106 cells or over a period of time. Source: Animal Cell Culture: A Guide to Basic Techniques, Fifth Edition.
Operation method
Program 21.6 Protein Synthesis Experiments Move makings For more product details, please visit Aladdin Scientific website.
non-sterile
Cell culture, e.g. 1X104 to 1X106 cells, 24-well plate
3H-leucine. 2 MBq /ml (~50uCi/ml) in blood-free chemonium (specific activity is not important as it will be determined by the concentration of leucine in the medium)
Non-sterile
SLS or SDS, 1% (35m mol/L ) dissolved in 0 .3 mol/L NaOH
Trichloroacetic acid (TCA)
Liquid Flash Bottle
Eppendorf tubes
Scintillation liquid, min. 10% water
Procedure
1. Grow cells to desired density.
2. Remove the plate from the incubator and add pre-warmed radioisotope dissolved in culture medium or BSS at a dilution of 1:10 (e.g. l00ul/ml/well).
3. Return the cells to the incubator as soon as possible.
4 . Continue incubation for 4 to 24 h.
TIP: Different proteins have different renewal rates. This procedure is not specific to any particular kind of protein, but can be used for total protein in rapidly growing cells. When first used to determine protein synthesis in a cell line, verify that the synthesis rate is linear over the selected incubation time. Delays can occur if the amino acid pool is lower than at saturation.
5 . Remove the culture from the incubator and carefully aspirate the culture fluid from the wells into an appropriate container of discarded radioactive liquid (see Section 7.7.2).
1. gently wash the cells with cold HBSS or D-PBSA.
Some monolayers, especially loosely attached continuous cell lines such as HeLa-S, may break apart during washing. If this happens, remove the isotope and add methanol, fix the monolayer, let it rest for lO min, carefully remove the methanol and let it dry.
7. Place the plate on ice, add 0.6mol/L TCA, and allow to stand at 4°C for lO min: then aspirate off any unadulterated precursor.
8. Repeat step 7 twice, but only for 5 min each time.
9 . Wash the culture with MeOH and dry the plate.
10. Add 0.5 ml of 0.3 mol/L NaOH containing 1% SLS and leave for 30 min at room temperature.
11. Mix the cultures in each well and transfer them to a liquid flash bottle.
12. Add 5 ml of scintillation solution and count in a scintillator.
Hint: Biodegradable scintillants (e.g. Ecoscint) are preferable to toluene or xylene based scintillation solutions as they are less toxic and can be poured into the sink with a large amount of water, as long as the radioactivity is below the prescribed amount. For suspension cultures, centrifuge the cells at 1000 g for l0 min in step 5 to remove the medium, and repeat this step except for step 9.6,7,8 The plates from step 9 can also be quantified directly on a phosphorimager.