Mar 11, 2024
Cell Lysate Preparation & Immunoblotting Protocols
- 1Department of Biology, Misericordia University
Protocol Citation: Caitlyn Henry 2024. Cell Lysate Preparation & Immunoblotting Protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3dwnl3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 10, 2024
Last Modified: March 11, 2024
Protocol Integer ID: 96460
Abstract
The purpose of this experiment is to investigate the effects of forskolin-mediated cAMP activation on NF-κB, TNF-ɑ, AKAP95, and cyclin D3 expression in 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 3 hours. Cell lysates were prepared, and immunoblotting was subsequently performed to quantify changes in NF-κB, TNF-ɑ, AKAP95, and cyclin D3 expression in response to the different treatment combinations.
Materials
To prepare RT4-D6P2T cell lysates:
- RT4-D6P2T Schwann cells (ATCC, Cat #CRL-2768, Manassas, VA)
- Dulbecco’s Modified Eagle Medium (DMEM) (ATCC, Cat #30-2002, Manassas, VA)
- 10% fetal bovine serum (FBS) (Thermo Fisher, Cat #16000044, Waltham, VA)
- 1% penicillin/streptomycin (Pen-strep) (GIBCO, Cat #15140-015, Gaithersburg, MD)/amphotericin B (R&D Systems, Cat #B23192, Minneapolis, MN)
- Poly-L-lysine hydrobromide (PLL) (Sigma, Cat #P9155, St. Louis, MO)
- Three (3) 6-well plates
- Hanks' Balanced Salt Solution (HBSS) (Sigma, Cat #55021C-1000ML, St. Louis, MO)
- N2 media: DMEM/F12, no phenol red (Thermo Fisher, Cat #21041025, Waltham, MA); insulin (Sigma, Cat #91077C, St. Louis, MO); and apo-transferrin (Sigma, Cat #T1147, St. Louis, MO)
- Forskolin (Sigma, Cat #F-6886, St. Louis, MO)
- Lipopolysaccharide (LPS) from Escherichia coli O111:B4 (Sigma, Cat #L2630, St. Louis, MO)
- IP lysis buffer (Thermo Fisher, Cat #87787, Waltham, MA)
- 10% 100X protease inhibitor cocktail (PIC) (Sigma, Cat #P8849, St. Louis, MO)
- PBS, pH 7.4 (1X) (Thermo Fisher, Cat #10010023, Waltham, MA)
- Cell scrapers (VWR, Cat #10062-904, Radnor, PA)
To run the gel:
- Ammonium persulfate (APS) (Sigma, Cat #A3678, St. Louis, MO)
- Bio-Rad Mini-PROTEAN Tetra Handcast System
- Resolving buffer (VWR, Cat #M197, Radnor, PA)
- TEMED
- Bisacryl (37.5:1) (VWR, Cat #M157, Radnor, PA)
- 0.01% SDS solution OR 10% isopropyl EtOH
- Cardstock paper
- 4X stacking buffer (VWR, Cat #M198, Radnor, PA)
- 1X running buffer
- Protein marker (Bio-Rad Laboratories, Cat #161-0375, Hercules, CA)
To prepare for transfer:
- PVDF membranes (Immobilon P – 0.45 μm, Cat #IPVH00010)
- 1X transfer buffer
- Bio-Rad Mini Trans-Blot Cell Transfer System
Western blotting:
- Blotto: 10X Tris/NaCl, DI H2O, Tween 20, and Blotto Instant Milk (or use TTBS as an alternative to Blotto)
- Anti-rat NF-κB p65 antibody (dilution – 1:750, Thermo Fisher, Cat #436700, Waltham, MA)
- Anti-TNF-α antibody (dilution – 1:1000, Thermo Fisher, Cat #BMS175, Waltham, MA)
- Anti-AKAP95 antibody (dilution – 1:2500, gifted by Vince Coghlan, OHSU, Portland, OR)
- Anti-cyclin D3 antibody (dilution – 1:1000, BD Biosciences, Franklin Lakes, NJ)
- Anti-actin antibody (dilution – 1:1000, Sigma-Aldrich, Cat #A2066, St. Louis, MO)
- SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher, Cat #34580, Waltham, MA)
- Stable peroxide solution
- Aluminum foil
- Binder sheet protectors
- Image Lab 6.1 software (Bio-Rad Laboratories, Hercules, CA)
To prepare RT4-D6P2T cell lysates (for three 6-well plates):
To prepare RT4-D6P2T cell lysates (for three 6-well plates):
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.
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.
Incubate cells in DMEM for 24 hours.
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.
Incubate cells in N2 media for 24 hours.
After 24 hours, prepare the forskolin-supplemented media by adding 10 μL of a 2 mM forskolin stock (Sigma, Cat #F-6886, St. Louis, MO) to 20 mL of N2 media.
Add 2 mL of the appropriate medium to each well following the plate layout (see example below).
After adding the media, add the appropriate LPS dose to each well following the plate layout.
0.1 μg/mL LPS: 2 μL of 100 μg/mL LPS stock OR 20 μL of 10 μg/mL LPS stock
1 μg/mL LPS: 2 μL of 1 mg/mL LPS stock OR 20 μL of 100 μg/mL LPS stock
10 μg/mL LPS: 20 μL of 1 mg/mL LPS stock
Allow cells to incubate in the different treatment combinations for the required incubation time (3 hours).
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.
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).
Wash each well 2x with non-sterile PBS.
After the last wash, add 100 μL prepared lysis buffer to each well.
Thoroughly scrape the surface of each well and pipette the lysates into the appropriately labeled Eppendorf tubes (on ice).
Agitate tubes at ~4°C for 30 minutes.
After 30 minutes, centrifuge the tubes at ~4°C and 10,000 RPM for 20-30 minutes (or until a pellet is visible).
After a pellet has formed in each tube, collect the supernatant.
Store lysates at -80°C for future use.
To run the gels (makes 2 small gels):
To run the gels (makes 2 small gels):
Dissolve ammonium persulfate (APS) (Sigma, Cat #A3678, St. Louis, MO) in DI H2O to prepare a 0.1 mg/mL solution.
Clean small glass plates (Bio-Rad), glass molds, rubber sponges, and combs with 70% EtOH and assemble gel plates.
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.
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.
Fill the plates with resolving gel immediately up to the mark made on the glass from step 21. 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.
Allow gel to polymerize for 15-25 minutes.
Once gel has fully polymerized, blot out the SDS/isopropyl EtOH with pieces of cardstock paper.
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).
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.
Allow gel to polymerize for 15-25 minutes.
Once gel has fully polymerized, disassemble gel apparatus and slowly remove comb from glass plates under running water.
Place gel assemblies into small box with the short glass plate facing inward and clamp into position.
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.
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.
Use Bio-Rad Powerpac supply to run the gel.
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.
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.
Continuously check buffer levels. If it runs low, replenish the top of the chamber with buffer using a 10 mL syringe.
To prepare for transfer:
To prepare for transfer:
Cut 4 pieces of cardboard the same size as the small glass plate (10 cm x 8.5 cm).
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.
Make 500 mL 1X transfer buffer.
Soak all sponges in 1X transfer buffer.
Once the gel has completed its run (you will notice that the dye front is at the bottom), turn off the electrical unit.
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.
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.
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.
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 the black surface and the white side facing the red surface.
Place ice pack in back of transfer box.
Fill the chamber with 1X transfer buffer, enough to cover the sponges and place a stir bar at the bottom.
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.
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.
Western blotting:
Western blotting:
Rehydrate membrane(s) in methanol, followed by two rinses in DI H2O, if necessary.
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.
Cut membrane into as many pieces as needed and soak membrane strips in Blotto on rocker for 1-1.5 hours.
Mix primary antibody following manufacturer's instructions.
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.
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.
Mix secondary antibody following manufacturer's instructions.
After the 5th wash, add secondary antibody to dishes and incubate for exactly 1 hour.
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.
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.
Cut sheet protector into the appropriate size for each membrane strip.
After the last wash, add luminol solution and incubate on a rocker for 1 minute in the dark.
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).
Analyze protein bands using Image Lab 6.1 software (Bio-Rad Laboratories, Hercules, CA). Follow instructions as specified for protein target.