Sample preparation for Immunobloting
1. Seed cells to 6 well-plate (1x106 cells per well) and incubate overnight at 370C, 5% CO2
2. Treat cells for …. Minute / Hours according to experimental condition
3. Wash the cell twice with 1x PBS (1mL)
4. Add 1mL PBS and pipette up and down vigorously to detach the cells from floor of the plate
5. Collect the cells in a 1.5 mL eppendorff tube preserved in an ice box
6. Spin at 10,000 RPM for 1 min and then remove supernatant
7. Add 150 µl lysis Buffer and mix well by pipetting up and down.
8. Keep eppendorff tube in ice box
9. Sonicate sample in each eppendorff tube for 30 time (one second pulse with 50% duty or power level 3.0)
10. keep in -200c for next day work
Protocol for SDS-PAGE and immunoblotting
Day-1
1. Heat sample for 5 minute at 1000c
2. Centrifuge at 10,000 rpm for 5 minutes at 40C
3. Set mini gel apparatus for SDS-PAGE
Ø Clean glass plates and alumina plates with ethanol.
Ø Assemble the glass plates and spacers.
Ø Mark a line 5.5cm from the bottom of a long side of the glass
Ø Attach the glass plate assembly to the holder.
Ø To prevent leak use parafilm at the bottom
4. Make 5 ml of separating gel (Tris-HCl ph8.8) depending on MW of protein (15% Acrylamyde is for LC3, 10% for Beclin1)
5. Add the separating gel with a pasteur pipette.
6. Gentlely add 3A water to the top of this until the level reaches the top of the plates.
7. The separating gel should polymerize in 30 minutes.
8. Make the stacking gel reagent (Tris-HCl ph6.8)
9. Pour off 3A water. Dry the watery interplate surface with a piece of Whatmann paper.
10. Insert the comb straight on down, then pour the stacking gel on top of the polymerized separating gel to fully seal the comb. Remove any bubbles from underneath the comb, if possible, by moving the comb gently from side to side so the bubbles get into the space in between and float up.
11. The stacking gel should polymerize in 20 to 30 minutes.
12. Add some sds page buffer in SDS-PAGE apparatus
13. Remove the glass-gel set from holder and set it in SDS-PAGE apparatus
14. Remove the comb from glass-gel set
15. Fill the space with SDS-PAGE buffer
16. Load 2µl marker into first well of the SDS-PAGE gel
17. Load 10 µl of sample into the well of the SDS-PAGE gel
18. Run gel for at 25 mA, run until the blue marker goes to the end of the gel (about 1 hr)
19. While the gel is running, prepare the transfer buffer, and cut out a piece of nitrocellulose membrane (9x6 cm) and four pieces of Whatmann filter paper for the transfer. Biorad tank takes about 2.5 liters of transfer buffer.
20. Cut out the Whatmann filter papers so they each are slightly larger than the gel in each dimension--about 1/4 to 1/2 cm larger. Cut out the nitrocellose so that it is slightly smaller than the Whatmann filter papers but still covers the entire surface of the gel.
21. Prewet the nitrocellulose membrane in TOWBIN’s buffer for at least five minutes before blotting step.
22. Start cooling machine and put magnetic stirrer in transfer tank to cool the it at 40C
23. Remove the plates from the apparatus. Remove the spacers and pry off one of the plates by bending the spacer. The gel should now be stuck to one of the plates.
24. Carefully float the gel off the plate into a tupperware container containing transfer buffer. .
25. When the gel has equilibrated, construct a "sandwich" for the transfer as follows:
a) Place some transfer buffer in a large glass or tupperware container and place the plastic frame inside so the white side is lying in the buffer and the black side is folded out.
b) Place a sponge on top of the white side, then stack two sheets of the Whatmann paper you cut out on top. These things should be submerged in the buffer so that they become completely wet.
c) Place the nitrocellulose filter, labeled appropriately, on top, then carefully position the gel on top of this.
d) Add two more pieces of Whatmann filter paper, then a second sponge. Then press everything down into the buffer and squeeze out any air bubbles that might be trapped in the sandwich using a pipette as a roller.
e) Close the sandwich by folding the black side over onto the white and locking it in place. Use rubber band to lock it. Then immerse the sandwich in transfer tank to which transfer buffer has been added. Since the proteins will migrate toward the positive end, make sure that the nitrocellulose side is closest to the positive electrode. Make sure that the lid is put on the right side. That is, the white side (membrane) of the sandwich should be facing the red side and the black side (gel) facing the black side
26. Electrotransfer to nitrocellulose membrane at 90 V for 1.5 hr. Make sure to have the cooling unit on at 4 C so that cold water can circulate through the tank while the transfer is taking place. Otherwise, if the buffer gets hot, bubbles of air may come out of solution and become trapped in the sandwich.
1. After transfer, stain the membrane with 1x ponceau S for 5 sec. and destain with ddH20
2. Wash membrane with 1x PBS for 5 min
3. Block the membrane for 1h with 5ml of 5% blocking reagent (Roche) in PBS on the shaker
4. Incubate with primary antibody (antibody in 5ml of 2.5% blocking reagent in PBS) for overnight, Incubate at 40C on the shaker
Day 2
5. Wash membrane 3 times (10 min. each time) with 10 ml of 0.1% Tween 20 in PBS at RT
6. Incubate with secondary antibody (antibody in 5ml of 2.5% blocking reagent in PBS) for 1hr at RT
7. Wash membrane 4 times (15 min. each time) with 10 ml of 0.1% Tween 20 in PBS at RT
8. Cut and set plastic bag in X-ray box during last wash
9. Detect protein with Enhanced Chemi-Luminescence (ECL). Incubate membrane in ECL solution (prepared fresh by mixing 20μL of ECL 1 in 2mL of ECL 2) for 2 min. Drain the ECL and wrap the membrane in plastic bag and expose to film.
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