TITLE: Cell Lysate Preparation &amp; Immunoblotting Protocols
AUTHORS: aasirvatham

[DESCRIPTION]
The purpose of this experiment is to investigate the effects of forskolin-mediated cAMP activation on the viability of LPS-treated Schwann cells. The immortalized rat RT4-D6P2T cell line (ATCC #CRL-2768) was cultured and received one of the following treatments: 0.1, 1, or 10 μg/mL of LPS, in N2 media (control) or N2 media supplemented with 2 μM of forskolin, for 1, 3, 12, or 24 hours. Cell lysates were prepared, and immunoblotting was performed to quantify changes in NF-κB, TNF-ɑ, AKAP95, and cyclin D3 expression in response to the different treatment combinations. 

[STEPS]
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
1. Aseptically culture immortalized rat RT4-D6P2T Schwann cells (ATCC, Cat #CRL-2768, Manassas, VA) in Dulbecco’s Modified Eagle Medium (DMEM) (ATCC, Cat #30-2002, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher, Cat #16000044, Waltham, VA) and 1% penicillin/streptomycin (Pen-strep) (GIBCO, Cat #15140-015, Gaithersburg, MD)/amphotericin B (R&D Systems, Cat #B23192, Minneapolis, MN) at 37°C and 5% CO2 in poly-L-lysine-coated dishes.

SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
2. At 80% confluency, split and seed cells into DMEM (2 mL DMEM/well) in three 6-well plates at a density of ~300,000 cells/well.
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
3. Incubate cells in DMEM for 24 hours.
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
4. After 24 hours, aspirate the DMEM and wash each well 2-3x with 2 mL HBSS. After the last wash, add 2 mL N2 media (DMEM/F12, no phenol red [Thermo Fisher, Cat #21041025, Waltham, MA] supplemented with 5 μg/mL insulin [Sigma, Cat #91077C, St. Louis, MO] and 100 μg/mL apo-transferrin [Sigma, Cat #T1147, St. Louis, MO]) to each well.
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
5. Incubate cells in N2 media for 24 hours.
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
6. After 24 hours, prepare the forskolin-supplemented media by adding 10 μL of a 2 mM forskolin stock to 20 mL of N2 media.
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
7. Add 2 mL of the appropriate medium to each well following the plate layout.
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
8. After adding the media, add the appropriate LPS dose to each well following the plate layout. For a 0.1 μg/mL dose of LPS, add 2 μL of a 100 μg/mL LPS stock OR 20 μL of a 10 μg/mL LPS stock. For a 1 μg/mL dose of LPS, add 2 μL of a 1 mg/mL LPS stock OR 20 μL of a 100 μg/mL LPS stock. For a 10 μg/mL dose of LPS, add 20 μL of a 1 mg/mL LPS stock. 
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
9. Allow cells to incubate in the different treatment combinations for the required incubation time (3 hours).
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
10. After the required incubation time, remove the 6-well plates from the incubator and immediately put plates on ice. Keep plates on ice for the following steps.
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
11. Make ~3 mL lysis buffer by adding 2.970 mL IP lysis buffer (Thermo Fisher, Cat #87787, Waltham, MA) to 30 μL 10% 100X protease inhibitor cocktail (PIC) (Sigma, Cat #P8849, St. Louis, MO).
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
12. Wash each well 2x with non-sterile PBS.
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
13. After the last wash, add 100 μL prepared lysis buffer to each well.
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
14. Thoroughly scrape the surface of each well and pipette the lysates into the appropriately labeled Eppendorf tubes (on ice).
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
15. Agitate tubes at ~4°C for 30 minutes.
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
16. After 30 minutes, centrifuge the tubes at ~4°C and 10,000 RPM for 20-30 minutes (or until a pellet is visible).
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
17. After a pellet has formed in each tube, collect the supernatant.
SECTION: To prepare RT4-D6P2T cell lysates (for three 6-well plates):
18. Store lysates at -80°C for future use.
SECTION: To run the gel (makes 2 small gels):
19. Dissolve ammonium persulfate (APS) (Sigma, Cat #A3678, St. Louis, MO) in DI H2O to prepare a 0.1 mg/mL solution.
SECTION: To run the gel (makes 2 small gels):
20. Clean small glass plates (Bio-Rad), glass molds, rubber sponges, and combs with 70% EtOH and assemble gel plates.
SECTION: To run the gel (makes 2 small gels):
21. Insert 10-well comb (Bio-Rad) into apparatus and make small mark with marker on the glass just below comb, to indicate the bottom of the wells.
SECTION: To run the gel (makes 2 small gels):
22. Make resolving gel consisting of 4.8 mL DI H2O, 2.5 mL resolving buffer (VWR, Cat #M197, Radnor, PA) premixed with TEMED at 10 μL/mL, 2.6 mL Bisacryl (37.5:1) (VWR, Cat #M157, Radnor, PA), and 75 μL APS.

SECTION: To run the gel (makes 2 small gels):
23. Fill the plates with resolving gel immediately up to the mark made on the glass from step 3. Add small amount of 0.01% SDS solution or 10% isopropyl EtOH to the top of resolving gel with a disposable pipette to prevent the gel from drying out.
SECTION: To run the gel (makes 2 small gels):
24. Allow gel to polymerize for 15-25 minutes.
SECTION: To run the gel (makes 2 small gels):
25. Once gel has fully polymerized, blot out the SDS/isopropyl EtOH with pieces of cardstock paper.
SECTION: To run the gel (makes 2 small gels):
26. Prepare stacking gel by adding 4.5 mL stacking buffer, 0.5 mL Bisacryl, and 5 μL TEMED. (To make stacking buffer, use 137.5 mL of a 4X stock [VWR, Cat #M198, Radnor, PA], 550 μL TEMED, and 362 mL DI H2O, and refrigerate).
SECTION: To run the gel (makes 2 small gels):
27. Add the prepared stacking gel on top of the resolving gel layer and place comb in immediately. Wipe off any gel spillage on the outside of the glass, if necessary.
SECTION: To run the gel (makes 2 small gels):
28. Allow gel to polymerize for 15-25 minutes.
SECTION: To run the gel (makes 2 small gels):
29. Once gel has fully polymerized, disassemble gel apparatus and slowly remove comb from glass plates under running water.
SECTION: To run the gel (makes 2 small gels):
30. Place gel assemblies into small box with the short glass plate facing inward and clamp into position.
SECTION: To run the gel (makes 2 small gels):
31. Add 1X running buffer to the box, taking care to ensure that the electrodes are fully submerged in buffer. Make sure that the inner chamber is filled to the brim with running buffer, and fill 1/3 of the gel box with running buffer.
SECTION: To run the gel (makes 2 small gels):
32. Add 4 μL marker (Bio-Rad Laboratories, Cat #161-0375, Hercules, CA) to designated gel walls and add 20 μL (maximum 25 μL) sample (RT4-D6P2T cell lysate) to designated gel wells with gel loading pipettes.
SECTION: To run the gel (makes 2 small gels):
33. Use Bio-Rad Powerpac supply to run the gel.
SECTION: To run the gel (makes 2 small gels):
34. Place lid on gel box and connect the leads to the power supply, taking care to see that the red electrode connects to the red terminal, and similarly with the black electrode.
SECTION: To run the gel (makes 2 small gels):
35. Turn on electricity to 95V for the first 15-20 minutes, and once marker is about halfway down the gel, turn electricity to 105V for 45 minutes. Total gel run may take up to 2 hours.
SECTION: To run the gel (makes 2 small gels):
36. Continuously check buffer levels. If it runs low, replenish the top of the chamber with buffer using a 10 mL syringe.
SECTION: To prepare for transfer:
37. Cut 4 pieces of cardboard the same size as the small glass plate (10 cm x 8.5 cm).
SECTION: To prepare for transfer:
38. Cut 2 PVDF membranes (Immobilon P – 0.45 μm, Cat #IPVH00010) to a dimension of 8.2 cm x 6 cm. Handle only the corners of the membrane with fingertips or forceps.
SECTION: To prepare for transfer:
39. Make 500 mL 1X transfer buffer.
SECTION: To prepare for transfer:
40. Soak all sponges in 1X transfer buffer.
SECTION: To prepare for transfer:
41. Once the gel has completed its run (you will notice that the dye front is at the bottom), turn off the electrical unit.
SECTION: To prepare for transfer:
42. Prepare the membrane by thoroughly soaking it in methanol followed by two rinses in DI H2O. Then place the membrane in 1X transfer buffer for 5 minutes on a rocker.
SECTION: To prepare for transfer:
43. Remove the gel from the cassette very slowly. With a flat spatula, remove the comb area of the gel and any folded area on the bottom below the dye front if necessary.
SECTION: To prepare for transfer:
44. In a Pyrex dish filled with 1X transfer buffer, assemble the sandwich as follows: black cassette down, sponge, cardboard, gel, membrane, cardboard, and sponge. Close and lock the sandwich.
SECTION: To prepare for transfer:
45. Place the whole assembly in the chamber with a stir bar in the bottom. Make sure that the cassette in the transfer box has the black side facing black and the white side facing the red surface.
SECTION: To prepare for transfer:
46. Place ice pack in back of transfer box.
SECTION: To prepare for transfer:
47. Fill the chamber with 1X transfer buffer, enough to cover the sponges and place a stir bar at the bottom.
SECTION: To prepare for transfer:
48. Place lid on transfer box, connect to the Bio-Rad power supply, and color match the electrodes with red on red and black on black. Transfer gel at 100V for exactly 1 hour.
SECTION: To prepare for transfer:
49. After completion, wash the sponges and cassette along with the chamber. Discard the gel and the cardboard piece but save the membrane. Mark the marker lines on the membrane with a pen and allow membrane to dry. Store membrane at room temperature.
SECTION: Western blotting:
50. Rehydrate membrane(s) in methanol, followed by two rinses in DI H2O, if necessary.
SECTION: Western blotting:
51. Make 1000 mL Blotto by combining 100 mL 10X Tris/NaCl, 800 mL DI H2O, 10 mL Tween 20, and 50 g Blotto Instant Milk.
SECTION: Western blotting:
52. Cut membrane into as many pieces as needed and soak membrane strips in Blotto on rocker for 1-1.5 hours.
SECTION: Western blotting:
53. Mix primary antibody following manufacturer's instructions.
SECTION: Western blotting:
54. After 1-1.5 hours of incubation in Blotto, remove Blotto from dishes, add primary antibody, and incubate on rocker for 1 hour at room temperature or overnight at ~4°C.
SECTION: Western blotting:
55. After 1-hour primary antibody incubation, perform five 5-minute washes (or three 10-minute washes). If the primary antibody incubation was in Blotto, use Blotto. If the primary antibody incubation was in TTBS, use TTBS.
SECTION: Western blotting:
56. Mix secondary antibody following manufacturer's instructions.
SECTION: Western blotting:
57. After the 5th wash, add secondary antibody to dishes and incubate for exactly 1 hour.
SECTION: Western blotting:
58. After 1-hour secondary antibody incubation, perform five 5-minute washes (or three 10-minute washes). If Blotto was used, perform two washes in Blotto, followed by three washes in TTBS (or one wash in Blotto, followed by two washes in TTBS). If TTBS was used, perform all washes using TTBS.
SECTION: Western blotting:
59. Mix equal amounts of SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher, Cat #34580, Waltham, MA) and stable peroxide solution in 15 mL conical wrapped in aluminum foil. These chemicals are light-sensitive.
SECTION: Western blotting:
60. Cut sheet protector into the appropriate size for each membrane strip.
SECTION: Western blotting:
61. After the last wash, add luminol solution and incubate on a rocker for 1 minute in the dark.
SECTION: Western blotting:
62. Place each membrane strip into a sheet protector and place a small drop of fluorescent paint onto the ink lines (which designates the marker bands).
SECTION: Western blotting:
63. Analyze protein bands using Image Lab 6.1 software (Bio-Rad Laboratories, Hercules, CA). Follow instructions as specified for protein target.