The samples were mixed with TruPAGE LDS Sample Buffer (Sigma-Aldrich) and electrophoresed in a 4–20% TruPAGE SDS PAGE Gel (Sigma-Aldrich). Proteins were transferred to 0.45 µm PVDF membranes by electroblotting overnight. After the transfer was completed, the membranes were incubated in 1X TBST with 5% (w/v) nonfat dry milk for an hour at room temp. Primary antibody dilutions were made in 1xTBST containing 5% milk as follows: anti-RIP924 (link) 1:5000, anti-6× His HRP Conjugate (Invitrogen) 1:2500, anti-MBP (Invitrogen) 1:2500. Membranes were incubated with each respective antibody dilution on a plate shaker at 4 °C overnight, followed by three, 10-min washes with 1× TBST at room temperature. Membranes were incubated with Peroxidase Conjugated Goat anti-Rabbit (H + L) antibody (Invitrogen) for one hour at room temperature followed by three additional 10-min washes with 1X TBST. The ProtoGlow ECL Detection Kit (National Diagnostics) and an Azure c400 gel imager (Azure Biosystems Inc.) was used to visualize the blots.
Trupage lds sample buffer
Manufactured by Merck Group
TruPAGE LDS sample buffer is a laboratory reagent used for preparing protein samples for electrophoresis. It is a Lithium Dodecyl Sulfate (LDS) based buffer that denatures and solubilizes proteins prior to separation on a polyacrylamide gel.
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Lab products found in correlation
Odyssey clx, by LI COR (3 mentions)
Irdye 680rd goat anti mouse igg, by LI COR (2 mentions)
Protoglow ecl detection kit, by National Diagnostics (1 mentions)
Anti mbp, by Thermo Fisher Scientific (1 mentions)
Azure c400 gel imager, by Azure Biosystems (1 mentions)
Ab9108, by Abcam (1 mentions)
Ab216773, by Abcam (1 mentions)
Immobilon fl membrane, by Merck Group (1 mentions)
Revert total protein stain, by LI COR (1 mentions)
Chameleon duo ladder, by LI COR (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Omnipage mini vertical protein electrophoresis system, by Cleaver Scientific (1 mentions)
Image studio lite 5, by LI COR (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
X ray film, by Fujifilm (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Irdye 800cw donkey anti goat igg, by LI COR (1 mentions)
6 protocols using trupage lds sample buffer
1
Protein Expression and Detection Workflow
2
Quantitative Western Blotting Protocol
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Quantitative Western blotting was performed based on the method of Schütz et al. (65 (link)). Briefly, strains were grown for 16 h at 30 °C, 180 rpm shaking and 4 × 107 cells (equivalent to an OD600 of 4) were pelleted, washed in water, and resuspended in 1 mL 2 M lithium acetate. Cells were incubated on ice for 5 min, pelleted, resuspended in 200 µL 0.4 M NaOH, and incubated on ice for an additional 5 min. Cells were then centrifuged and resuspended in 100 µL TruPAGE LDS Sample Buffer (Sigma-Aldrich) containing 50 mM DTT and incubated at room temperature for 15 min.
Proteins were separated on a TruPAGE 10% polyacrylamide gel (Sigma-Aldrich) alongside the Chameleon Duo ladder (LI-COR) and transferred onto Immobilon-FL membrane (Merck Millipore). Total protein staining was performed using REVERT Total Protein Stain (LI-COR) following the manufacturer’s instructions.
The membrane was washed in PBS and blocked overnight in 0.1% Alkali-soluble Casein in 0.2× PBS. The membrane was then washed in PBS, incubated in Rabbit Anti-6X His tag antibody (ab9108; Abcam) (1:1,000) in blocking buffer +0.1% Tween20 for 1 h, washed four times in PBST, then incubated in Goat anti-Rabbit antibody conjugated to 800CW (ab216773; Abcam) (1:10,000) in blocking buffer +0.1% Tween20 for 3 h. The membrane was washed four times in PBST, then once in PBS before imaging on a LI-COR Odyssey Fc imaging system.
Proteins were separated on a TruPAGE 10% polyacrylamide gel (Sigma-Aldrich) alongside the Chameleon Duo ladder (LI-COR) and transferred onto Immobilon-FL membrane (Merck Millipore). Total protein staining was performed using REVERT Total Protein Stain (LI-COR) following the manufacturer’s instructions.
The membrane was washed in PBS and blocked overnight in 0.1% Alkali-soluble Casein in 0.2× PBS. The membrane was then washed in PBS, incubated in Rabbit Anti-6X His tag antibody (ab9108; Abcam) (1:1,000) in blocking buffer +0.1% Tween20 for 1 h, washed four times in PBST, then incubated in Goat anti-Rabbit antibody conjugated to 800CW (ab216773; Abcam) (1:10,000) in blocking buffer +0.1% Tween20 for 3 h. The membrane was washed four times in PBST, then once in PBS before imaging on a LI-COR Odyssey Fc imaging system.
3
Western Blot Protein Analysis Protocol
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Cells were lysed in SDS buffer [2% SDS, 50 mM NaCl, 50 mM Tris-HCl pH 7.4, 1× cOmplete™ protease inhibitor cocktail (Roche Diagnostics)] before boiling in TruPAGE™ LDS sample buffer (Sigma-Aldrich) supplemented with 0.1 M DTT for 10 min. SDS-PAGE was performed on hand-cast or TruPAGE™ SDS-polyacrylamide gels with TruPAGE™ TEA-Tricine SDS running buffer (Sigma) using the omniPAGE Mini Vertical Protein Electrophoresis System (Cleaver Scientific, Rugby, UK). Following transfer of proteins using a Mini-PROTEAN 2-D Electrophoresis Cell (Bio-Rad, Hercules, CA) to Immobilon®-P PVDF membranes (Merck-Millipore, Watford, UK) for chemiluminescence detection or for fluorescence detection, membranes were blocked with 5% dried skimmed milk powder in TBS-T. Incubations with primary antibodies (1:1000 dilution, except anti-SEC22B, 1:500, and anti-actin, 1:6000) were carried out overnight at 4°C, with appropriate horseradish peroxidase- or IRDye-conjugated secondary antibodies (1:1000 dilution) applied subsequently for 1 h at room temperature. Fluorescent detection of bound antibody was carried out using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) and X-ray film (Fujifilm), or by scanning with an Odyssey® CLx (LI-COR Biosciences, Lincoln, NE), followed by immunoblot quantification using Image Studio Lite 5.2.5 (LI-COR).
4
Tissue Lysis and Protein Extraction
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Tissue from each corpus callosum and V-SVZ were harvested and lysed with 20 mM Tris (pH 7.4), 1% sodium dodecyl sulfate and 1:50 dilution of protease and phosphatase inhibitor cocktails (Set II, Calibiochem) at 95°C for 15 min, with trituration at 10 min. Lysates were then centrifuged at 16,110 g for 10 min on a countertop centrifuge to remove insoluble material. Protein concentration was determined using a Bradford protein assay (Bio-Rad). Tissue lysates were prepared for SDS-PAGE with TruPAGE LDS sample buffer (Sigma) with 12% β-mercaptoethanol (Sigma).
5
Quantifying OmpH Protein Expression in CEF Cells
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The expression of OmpH protein in recombinant virus-infected CEF cells was determined by Western blot analysis using mouse polyclonal anti-OmpH as modified from the method as described previously [36 (link),37 (link)]. Briefly, 2.5 × 106 primary CEF were seeded into T25 flask the day before infection. The following day, the parental virus and each recombinant virus were diluted with growth medium to MOI 0.01 and added to the flask were then incubated at 38.5 °C and 5% CO2. At 48 h post infection, more cytopathic effects (CPE) were shown. The infected CEF cells were collected and boiled with TruPAGE LDS sample buffer (Sigma) for 7 min. The samples were separated on a 4–12% TruPAGETM Precast Gel, and the resolved proteins were transferred onto PVDF membranes. Immunoblots were blocked with 5% skimmed milk, then incubated with anti-OmpH primary antibodies (1:5000 dilution). After probing with primary antibodies, the blots were incubated with secondary antibody IRDye680RD goat anti-mouse IgG (LI-COR) and visualized using Odyssey Clx (LI-COR). On the other hand, duck serum positive DEV was used as primary antibody for DEV loading control using the same strategy.
6
Western Blot Analysis of Meq, Pu.1, v-rel, and GFP
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Approximately 1 × 106 HP8-ΔmiR-M4 cells and the control cells were collected and boiled with TruPAGE LDS sample buffer (Sigma) for 10 min. The samples were separated on a 4% to 12% TruPAGE precast gel, and the resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. Expression levels of Meq, Pu.1, v-rel, and GFP were detected using anti-Meq monoclonal antibody (MAb) FD7 (21 (link)), rabbit anti-SPIB polyclonal antibody (Aviva Systems Biology), anti-v-rel MAb HY87 (35 (link)), and GFP polyclonal antibody (SICGEN), respectively. The loading control used in all cases was α-tubulin (Sigma-Aldrich). After probing with primary antibodies was performed, the blots were incubated with secondary antibody IRDye®680RD goat anti-mouse IgG (Li-Cor) (for detection of Meq, v-rel, and α-tubulin), IRDye®800CW donkey anti-rabbit IgG (Li-Cor) (for detection of Pu.1), and IRDye®800CW donkey anti-goat IgG (Li-Cor) (for detection of GFP), and the results were visualized using Odyssey Clx (Li-Cor). For GFP detection, the PVDF membrane used for v-rel detection was stripped and reprobed with GFP antibody following the same procedure.
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