Mini protean tgx stain free protein gel

A total of 30 μg of each spinal cord sample was denatured using 2× Laemmli buffer containing 5% β-mercaptoethanol at 95 °C for 5 min and loaded onto a Mini-PROTEAN^® TGX Stain-Free™ Protein Gel (Bio-Rad, Hercules, CA, USA). All gels ran for 15 min at 80 V, followed by an additional 30 min at 200 V. Following separation, the samples were transferred onto a polyvinylidene difluoride membrane (Bio-Rad) using the Trans-Blot^® Turbo™ machine (Bio-Rad). After transfer, membranes were incubated with primary antibody against the 5-HT_2A receptor (rabbit, 1:500, Immunostar, Hudson, WI, USA) and glyceraldehye-3-phosphate dehydrogenase (GAPDH, mouse, 1:2000, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Membranes were then probed with an anti-rabbit or mouse HRP-linked secondary antibody (1:10,000, Cell Signaling). Membranes were then developed with the Clarity™ Western ECL substrate kit (Bio-Rad) and imaged using HyBlot CL™ autoradiography film (Denville, Metuchen, NJ, USA). Receptor expression was normalized to the corresponding protein density of GAPDH. Following normalization, data was averaged and further compared between naïve and SCI groups with a student’s t-test (p < 0.05).

A Protease K (PK) solution at 50 μg/mL (IBI Scientific, Dubuque, IA) in digestion buffer (10 mM Tris, pH 8.0, 2 mM CaCl₂) was diluted (0.1 eq.) into 30 μL of PMCA solution (with or without NS132) and incubated for 30 min at 37 °C. Subsequently, the samples were diluted (1:1, v/v) in SDS Protein Gel Loading Dye 2× (Quality Biological, Gaithersburg, MD) and loaded on Mini-PROTEAN TGX Stain-Free Protein Gel (BioRad, Hercules, CA). The gel was stained using the Fairbanks staining method and imaged using ChemiDoc MP (BioRad, Hercules, CA).

Purified protein fractions were separated on a Mini-PROTEAN TGX Stain-Free protein gel (Bio-Rad Laboratories, Inc.). Separated protein bands were visualized using “Stain Free Gel” application mode on ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc.). Protein gel was transferred to a nitrocellulose membrane (Bio-Rad, cat. #1704271) using a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, Inc.). Membranes were blocked in PBST containing 5% milk for 30 min at 22 °C. The membranes were then incubated with primary antibodies for GFP (custom anti-GFP rabbit polyclonal (provided by Foley lab, Memorial Sloan Kettering Cancer Center) at a dilution of 1:5,000) and His (mouse anti-penta-His antibody (Qiagen, cat. #34660) at a dilution of 1:10,000) in PBST + 5% milk overnight at 4 °C. The membranes were washed three times with PBST and were incubated with goat anti-rabbit IgG polyclonal antibody (IRDye 800CW (LI-COR Biosciences cat. #925-32211) at dilution of 1:10,000) and goat anti-mouse IgG polyclonal antibody (IRDye 680RD, LI-COR Biosciences #926-68070 at a dilution of 1:10,000) as the secondary antibodies in PBST + 5% milk for 1 hr at 22° C. The membranes were washed three times with PBST and imaged using a LI-COR (LI-COR Biosciences) and analyzed by ImageJ⁷⁵ .

Samples (25 μg each) were denatured and reduced in Laemmli 4x buffer (GTX16355, GeneTex) and 8% 2-mercaptoethanol for 5 mins at 95°C. Samples were then loaded onto either a 4–20% Mini-PROTEAN^® TGX Stain-Free™ Protein Gel (4568094, Bio-Rad) or 4–20% Mini-PROTEAN^® TGX™ Precast Protein Gel (4561094, Bio-Rad), along with a molecular weight marker (GTX49384, GeneTex). SDS-PAGE was carried out for 1 h at 100 V in 1x Running buffer (pH 8.3) using a Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad). Stain-Free Protein gels were checked for complete protein separation using the ChemiDoc Touch Imaging System (Bio-Rad).

As described in our previous publication [4 (link)], equal amounts of protein, measured by a Micro BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA), were mixed with 6 × SDS reducing sample buffer and boiled for 5 min before loading. The proteins (50 μg/lane) were separated on a 4–15% Mini-PROTEAN^® TGX Stain-Free™ Protein Gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred electrophoretically to 0.2 μm PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% nonfat milk in TTBS for 1 h at room temperature and incubated with primary antibodies against human Beclin1, ATG5, ATG7, LC3B, P62/SQSTM1 (Novus Biologicals, Littleton, CO, USA), or β-Actin (Boster Biological Technology, Pleasanton, CA, USA) overnight at 4 °C. After five washes with TTBS for 5 min each, the membranes were incubated with HRP conjugated Goat anti-Rabbit IgG (H+L) Secondary Antibody (1:10,000, Thermo Fisher Scientific, Rockford, IL, USA) or Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody (1:10,000, Thermo Fisher Scientific, Rockford, IL, USA) for 1 h at room temperature. The signals were detected with a Clarity™ Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA), and the images were obtained by a ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).

To evaluate the correct molecular weight of the different fusion proteins, the supernatant of transfected HEK293T cells was run with β-mercaptoethanol (Sigma-Aldrich, #63689) on a 4-20% Mini-Protean® TGX Stain-Free™ Protein Gel (BioRad, #4568093). After transfer to PVDF membranes (ThermoFisher, #88518), the membranes were blocked with 1x Pierce™ Clear Milk Blocking Buffer (ThermoFisher, #37587) and incubated overnight with Mouse anti-Human IgG (CH3 domain) Secondary Antibody (Invitrogen, #MA5-16557). After washing with TBS-Tween (ThermoFisher, #28360), the membranes were incubated for 45 min with Anti-mouse IgG HRP-conjugated (R&D systems, #HAF007) and developed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (ThermoFisher, #A38555). Images were obtained using the iBright FL1500 Imaging System (Invitrogen, #A44241) and analyzed with iBright Analysis Software Version 5.1.0 (Invitrogen).

Forty micrograms of proteins were separated a 4–15% Mini-PROTEAN™ TGX Stain-Free™ Protein gel (Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad, 1704271) using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). After saturation with 5% skimmed-milk in 1X Tris-Buffered Saline (TBS) buffer (blocking buffer) for 2H, membranes were incubated with primary antibody, anti-RAGE polyclonal goat IgG (1 : 5000 dilution in blocking buffer supplemented with 0.1% Tween-20) (Biotechne, R&D Systems France, AF1179), overnight at 4°C. Membranes were washed three times for 10 min with 1X TBS-0.1% Tween-20 (TBST) and incubated with a 1 : 5000 dilution of peroxidase-conjugated polyclonal anti-IgG goat (Abliance, BI2403) for 2 h. Blots were washed with TBST on three occasions for 10 min, rinsed with 1X TBS, and developed with the Clarity Max™ Western ECL Blotting Substrates (Bio-Rad, 1705062) according to the manufacturer's protocols. The All Blue Standard (Bio-Rad, 161-0373) was used as a protein ladder. The relative intensities of protein bands were analyzed using Image Lab™ software (BIO-RAD), and the results were presented as a ratio between the protein of interest and the total protein on the same blot. The use of stain-free imaging allows for the normalization of bands to the total protein on a blot, without requesting the use of housekeeping proteins or stripping and reprobing.