Adenovirus
Adenovirus Protocol
1. Reconstitute the plasmid in water
2. Add 0.5ul of plasmid to 10ul of bacteria
3. Let sit on ice for 20 minutes
4. Heat shock in water bath for 30 seconds
5. Put back on ice for a few minutes
6. Add 0.5ml of broth (use fire to keep sterile) (no antibiotic) and shake on heat block for 45 min
7. Plate 50ul onto one plate and 150ul onto another
8. Incubate overnight
9. Pick one colony from each plate and place in a tube of 2ml broth (one tube for each plate)
10. Put in shaker overnight at 37 ® C
11. If its cloudy in the morning you have lots of bacteria
12. Extract the plasmid DNA:
- a. Take 1.5ml from the culture tube and place in Eppendorf
- b. Centrifuge at 6000xg for 5 minutes at 4® C
- c. Pour off the supernatant
- d. Resuspend the pellet in 150ul of buffer P1 (from Qiagen Kit)
- e. Add 150ul of Buffer P2 and mix by inverting- should turn blue
- f. Incubate at room temperature for 5 min
- g. Add 150ul of cold buffer P3 and mix by inverting – should turn clear again and create a precipitate
- h. Incubate on ice for 5 minutes
- i. Centrifuge at 18000xg for 5 min at 4® C
- j. Pour off supernatant
- k. Elute DNA with 100% ethanol and centrifuge at 18000xg for 5 min at 4C
- l. Pour off as much as the ethanol as you can
- m. Wash the pellet with 70% ethanol and take off as much as you can
- n. Air dry the pellet and resuspend in 50ul of TE buffer
GSK3b Adenovirus Experiments
Neonatal Rat Cardiomyocytes Experiments
Infection with AV:
The titer of the AVs should be as follows:
*WT= 5X1010 pts/mL *F = 7 X 1010 pts/mL *E = 7.5 X 109 pts/mL
They are stored in the -80
For the previous experiments I was adding the following to my cells (Per 1 million cells)
*WT = 0.804 ul
*F = 0.125 ul
*E= 0.14 ul
Infect for 24 hrs and harvest in cell lysis buffer after washing twice.
Important:
- You do not need isolate the myofilament for the samples you’re using for western blot.
- You DO need to skin the samples that you’re using for the IP, because we want to isolate the myofilament. Therefore, DO NOT sonicate right after harvest. You need to wait until after you’ve skinned and resuspended in urea (protocol is below)
- You will need Empty, WT, F, E- with some wells/plates for western and some for IP
Western Blot
*TESTS TO MAKE SURE THAT THE VIRUS WORKED AND THAT OVEREXPRESSION IS EQUAL:
1. Harvest in cell lysis buffer (with inhibitors 1:100)- sonicate a few times in the cold room
2. Spin down 10 minutes @ 18.0 RPM in cold centrifuge
3. Use the supernatant for your western
4. I run two gels, loading about 10-15 uls of sample, you can nanodrop to get a quant but they’re usually pretty even
5. On one blot, probe with 1:2000 of anti-myc tag and 1:2000 ul of anti-flag tag, and on the other blot probe with 1:2000 gsk3b and 1:10,000 actin
6. After imagine, the blot of your tags should show that there is myc tag in the wild type, Y216F, and Y216E. There should be flag tag in Y216F and Y216E.
7. You should have 2 bands of GSK3b in your virus samples. The bottom band is endogenous and the top band is overexpressed. Quantify both bands individually in Image Studio and then calculate the amount of overexpression you’re getting. The goal is to have the amount of overexpression between samples be relatively even.
Co-Immunoprecipitation
*USED TO SEE IF BINDING DIFFERS BETWEEN MUTANTS: Prior to running the coIP you need to skin the cells.
Materials needed to skin:
- Standard relax buffer (SRB) - we keep a stock of this in the refrigerator.
- You need to make a stock to add 0.3% triton to (for skinning) and a stock with no triton (for washing).
- You need to add protease and phosphatase inhibitors to both of these (1:100).
- Read the below protocol to determine how much of each you need (depends on how many samples you have).
| 1mL | 2mL | 3mL | 5mL | |
|---|---|---|---|---|
| Urea 9 M (MW: 60.06) | 0.54g | 1.081g | 1.623g | 2.70g |
| Mixed Bed Resin | 0.1g | 0.2g | 0.3g | 0.5g |
1. Measure out urea and add MQH2O to an approximate volume (95% of total volume)
2. Leave on rotation until dissolved (30-40 min or ON), then adjust to the exact volume
3. Add Mixed Bed Resin, rotation for >10 min
4. Filter through 0.45μ filter
Sample prep (Skinning):
1. Spin down samples for 1 min @ 18.0 RPM in cold centrifuge
2. Pour off supernatant and add 1 ml of SRB with triton
3. Triturate the pellet gently and let sit on ice for 20 minutes
4. spin down for 1 min @ 18.0 RPM
5. Pour off supernatant and add 1 ml of SRB without triton
6. Triturate the pellet
7. Spin down again for 1 min @ 18.0 RPM
8. Pour off supernatant and add 1 ml of SRB without triton
9. Triturate the pellet
10. Spin down again for 1 min @ 18.0 RPM
11. Pour off the supernatant and re-suspend in 200 ul of 8M urea
12. Sonicate in cold room and then spin down for 10 min @ 18 RPM
13. Use the supernatant for your experiment.
Co-IP
- You can follow the coIP protocol for this part of the experiment, with the following specifications:
1. You are coIPing your control, wild type, Y216F, and Y216E with the myc tag antibody
2. Use all of the sample you skinned, aside from if you had to take some out for westerns. Dilute it with PBS-T so that the final volume of your protein is 200ul.
3. Use 10ul of myc tag antibody for each sample, dilute with PBS-T so that your final antibody volume is 200 ul
4. Incubate your protein with the antibody-bound beads for 10 minutes on hot dog roller
5. Make sure you keep your flow throw after incubating your protein with the antibody
6. Wash your beads after protein incubation 5 times with PBS-T
7. Add 60 ul of blue loading dye and 6 of reducing agent before boiling
8. Run your samples out on a gel with the coIPs on one side and the flow through on the other. Make sure to include an input.
9. Silverstain using the BioRad kit and image
