1. 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)
2. Soncate for 10-15 sec each on 30% (except for GSIS
3. Centrifuge @ 12000xg, 4C, 10 min (this step doesn't apply to GSIS)
4. Dilute 3ul samples into 27ul ddh20
5. Mix Reagents 50:1 (A:B)
6. Rinse plate
7. 190ul reagent mix +10ul samples (the order you add will be TBD by Amanda)
- Prepare samples in duplicate
8. Add 10ul for you standards
9. Incubate for a minimum of 30 min (up to 2 hours
10. 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)
1. Plate cells on 24 well plate, normally for 24/48/72/96 hours (palmitate group uses 3
plates per time period).
2. Must be at least 80% confluent per well.
3. Aspirate plate, label cells with 3H by adding 500ul per well of (0.25ul Thymidine per 1ml
of normal media).
4. Incubate for 15 minutes (run calculations for PBS, TCA & NaOH).
5. Dump 3H media into radioactive waste basket.
6. Wash each well with 200ul 1X PBS, three times. Dump after each wash.
7. Dump last of PBS, add 500ul 10% TCA and incubate plate on ice for 30 minutes.
8. Dump TCA, add 200ul of 0.3M normal NaOH per well.
9. STOP HERE -> Apply parafilm, freeze overnight.
10. 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.
1. Thaw plate from Radioactive Thymidine Assay at room temperature.
2. Thaw BCA standards A-I (9 total) over ice. (30-45min).
3. Label each scintillation tube, then add 50ul of plated cells to each corresponding tube.
4. Add one pump of scintillation fluid per scintillation tube.
5. Make mixture of BCA Protein Assay Standards, 1 part A to 50 parts B (1:51).
6. 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 B
7. 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.
8. Add 10ul of each standard to each well reserved for standards.
9. 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
10. Run BCA in Dr. Kenealey's lab next door (~5min)