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BCA Reference
BCA
- Harvest sample into Rippa + Protease solution (big dishes – 100ul, combinations of wells from a plate – 50ul) (Protease breaks up the proteins into amino acides, measures total amount of amino acids)
- Soncate for 10-15 sec each on 30% (except for GSIS
- Centrifuge @ 12000xg, 4C, 10 min (this step doesn't apply to GSIS)
- Dilute 3ul samples into 27ul ddh20
- Mix Reagents 50:1 (A:B)
- Rinse plate
- 190ul reagent mix +10ul samples (the order you add will be TBD by Amanda)
- Prepare samples in duplicate
- Add 10ul for you standards
- Incubate for a minimum of 30 min (up to 2 hours
- Read in plate reader
Radioactive Thymidine Assay
Thing you’ll need before starting:
- At least an hour of time (15-30min + 45min wait time)
- Sufficient 10% TCA (kept in a big clear bottle in the radioactive fridge)
- Sufficient 1X PBS
- Sufficient 0.3M normal NaOH
- Radioactive waste basket (kept in the cold room)
Procedure:
- Plate cells on 24 well plate, normally for 24/48/72/96 hours (palmitate group uses 3 plates per time period).
- Must be at least 80% confluent per well.
- Aspirate plate, label cells with 3H by adding 500ul per well of (0.25ul Thymidine per 1ml of normal media).
- Incubate for 15 minutes (run calculations for PBS, TCA & NaOH).
- Dump 3H media into radioactive waste basket.
- Wash each well with 200ul 1X PBS, three times. Dump after each wash.
- Dump last of PBS, add 500ul 10% TCA and incubate plate on ice for 30 minutes.
- Dump TCA, add 200ul of 0.3M normal NaOH per well.
- STOP HERE -> Apply parafilm, freeze overnight.
- Run Radioactive Thymidine BCA (shown below).
Radioactive Thymidine BCA
Things you’ll need before starting:
- At least 1-1.5 hours (the scintillation counter takes ~7 hours for 72 vials)
- Sufficient BCA standards (kept in left upper freezer)
- Sufficient BCA Protein Assay Standards A and B (kept around radioactive desk)
- Email lab and check dark room to verify no one else will be using the scintillation counter at the same time as you. You could attach a sticky note to the scintillation counter to let other labs know when you'll be using it as well.
Procedure:
- Thaw plate from Radioactive Thymidine Assay at room temperature.
- Thaw BCA standards A-I (9 total) over ice. (30-45min).
- Label each scintillation tube, then add 50ul of plated cells to each corresponding tube.
- Add one pump of scintillation fluid per scintillation tube.
- Make mixture of BCA Protein Assay Standards, 1 part A to 50 parts B (1:51).
- Add 150ul of mixture to each well.
- 72 wells + 9 standards = 81*150ul = 12,150ul total
- 12,150ul/51 = 238.24ul of A
- 238.24ul*50 = 11,911.77ul of B7.
- Add 10ul of each sample scintillation tube to each well (in the order you’ve organized to keep track of treatments). Reserve the first nine wells for standards A-I.
- Add 10ul of each standard to each well reserved for standards.
- Run scintillation counter in dark room. (~7hrs)
- Start code must be 8.
- Make sure no scintillation fluid is on vials
- 5min per vial (3 standard vials also in machine)
- When done save file on flash drive as .CSV
- Run BCA in Dr. Kenealey's lab next door (~5min)