Lysates (20 µg protein/lane) were separated by electrophoresis on SDS PAGE gels and transferred to polyvinylidene difluoride membranes, using Biorad Miniprotean system (BioRad). Blots were blocked and then incubated with antibodies against TP53 tumor suppressor protein (p53), B cell lymphoma 2 (BCL-2), and BCL-2 associated X protein (BAX) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), then further washed and incubated with corresponding secondary peroxidase-linked antibodies (Santa Cruz Biotechnology). Proteins were detected using Supersignal West FemtoChemiluminiscent substrate (Thermo Fisher Scientific, Rockford, IL, USA), and a Gel Doc Imaging system equipped with an XRS camera and Quantity One analysis software Image Lab 6.0 Biorad Laboratories). βactin (Sigma Aldrich) was used as a protein loading control.
Biorad miniprotean system
Manufactured by Bio-Rad
Sourced in United States
The Bio-Rad Mini-PROTEAN system is a compact and versatile electrophoresis system designed for running polyacrylamide gel electrophoresis (PAGE) of proteins and nucleic acids. It features a modular design that allows for the separation of up to two gels simultaneously. The system provides a consistent and reliable platform for various electrophoresis applications.
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Lab products found in correlation
Supersignal west femto chemiluminiscent substrate, by Thermo Fisher Scientific (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Secondary peroxidase linked antibodies, by Santa Cruz Biotechnology (3 mentions)
Gel doc imaging system, by Bio-Rad (3 mentions)
Quantity one analysis software, by Bio-Rad (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Tla 55, by Beckman Coulter (2 mentions)
Image lab analysis software, by Bio-Rad (2 mentions)
Image lab 6, by Bio-Rad (1 mentions)
β actin, by Merck Group (1 mentions)
Rabbit monoclonal primary antibodies, by Cell Signaling Technology (1 mentions)
Ecl substrate, by Merck Group (1 mentions)
Semi dry transfer system, by Bio-Rad (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Bca reagent, by Keygen Biotech (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ripa buffer, by Keygen Biotech (1 mentions)
Sds page gel kit, by Solarbio (1 mentions)
11 protocols using biorad miniprotean system
1
Western Blot Analysis of Apoptosis Regulators
2
Western Blot Analysis of PTEN/Akt Pathway
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RIPA buffer (KeyGen Biotech. Co., Ltd.) was used to collect total protein. Protein concentration was determined by BCA reagent (KeyGen Biotech). Spacer gel (5%) and 10% separation gel were prepared using an SDS-PAGE gel kit (Beijing, Solarbio Science & Technology Co., Ltd.). An equal amount of each protein (30 µg) was loaded to the gel and electrophoresis was performed on a Bio-Rad Mini-PROTEAN system (Bio-Rad Laboratories, Inc.). Proteins were transferred to 0.45 µm PVDF membranes (EMD Millipore) using a semi-dry transfer system (Bio-Rad Laboratories, Inc.). Membranes were blocked with 5% BSA at room temperature for 1 h and incubated with the following rabbit monoclonal primary antibodies (Cell Signaling Technology, Inc.) at 4°C overnight: PTEN (1:1,000; product no. 9188), Akt (1:1,000; product no. 4691), p-Akt (1:1,000; Ser473; product no. 4060) and β-actin (1:3,000; product no. 4970). After membranes were incubated with an HRP-conjugated secondary antibody (1:3,000; cat. no. sc-2357; Santa Cruz Biotechnology, Inc.) for 45 min at room temperature, blotting signals were visualized with ECL substrates (EMD Millipore). Densitometric analysis was conducted using Quantity One software (version 4.6; Bio-Rad Laboratories) and protein expression was normalized to endogenous β-actin.
3
Quantification of MAPK and NF-κB Signaling
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Total p44/42 MAPK (ERK 1/2) protein level was evaluated by Path Scan Sandwich ELISA tests according to the manufacturer's protocol (Cell Signaling Technology Inc.). Results are expressed in terms of OD units/mg protein of different treatment samples. NF-κB, phospho-NF-κB, and iNOS quantification were performed by Western blot technique.
Lysates (20 μg protein/lane) were separated by electrophoresis on 8% SDS PAGE gels under reducing conditions, then transferred to polyvinylidenedifluoride membranes (BioRad), using Biorad Miniprotean system (BioRad). Blots were then blocked and incubated with antibodies NOS2 (iNOS), NF-κB, and phospho-NF-κB (Ser536) (93H1) (Santa Cruz Biotechnology, Heidelberg, Germany) diluted in 1 : 500. After washing, the blots were incubated with corresponding secondary HRP-linked antibodies (1 : 1500) (Santa Cruz Biotechnology). Proteins were visualized and detected using Supersignal West Femto Chemiluminiscent substrate (Thermo Fisher Scientific, Rockford IL, USA) and a Gel Doc Imaging system equipped with a XRS camera and Quantity One analysis software (Biorad). GAPDH was used as a protein loading control.
Lysates (20 μg protein/lane) were separated by electrophoresis on 8% SDS PAGE gels under reducing conditions, then transferred to polyvinylidenedifluoride membranes (BioRad), using Biorad Miniprotean system (BioRad). Blots were then blocked and incubated with antibodies NOS2 (iNOS), NF-κB, and phospho-NF-κB (Ser536) (93H1) (Santa Cruz Biotechnology, Heidelberg, Germany) diluted in 1 : 500. After washing, the blots were incubated with corresponding secondary HRP-linked antibodies (1 : 1500) (Santa Cruz Biotechnology). Proteins were visualized and detected using Supersignal West Femto Chemiluminiscent substrate (Thermo Fisher Scientific, Rockford IL, USA) and a Gel Doc Imaging system equipped with a XRS camera and Quantity One analysis software (Biorad). GAPDH was used as a protein loading control.
4
Collagen Purity and Molecular Weight Analysis
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To evaluate the purity of the extracts and the molecular weight of the obtained protein fractions, a Bio-Rad Mini-PROTEAN® system (Bio-Rad, United States) was employed to separate proteins by SDS-PAGE. The lyophilized collagen samples were dissolved in 0.5 M acetic acid (2 mg/ml), mixed in a 1:1 (v:v) dilution with Laemmi sample buffer containing 5% (v/v) ß-mercaptoethanol and heated for 10 min at 70°C to denature the proteins. The 7.5% Mini-PROTEAN® TGX™ Precast Protein Gel (12-well, 20 μL) was loaded with 10 μL protein ladder and 10 μL from each collagen sample. The samples were run at 200 V for 30 min, and the gel was then stained using the Coomassie stain for 1 h with continuous stirring. Finally, the gel was detained in distilled water overnight and imaged using a 48-megapixel RGB camera.
5
TDP-43 Knockout in HeLa Cells
6
TDP-43 Knockout in HeLa Cells
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HeLa cells that were transfected with (1) Cas9 plasmid with or without the two guide RNAs to knockout TARDBP and (2) pEF06R plasmid were collected 9 days after transfection. Cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP −40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate), sonicated in 5 pulses and cellular debris was pelleted at 55,000RPM at 4°C for 30min in TLA55 (Beckman Coulter Inc). Protein concentration was determined using Pierce BCA Protein Assay kit (Thermo Fisher Scientific) according to the kit. Equal amounts of protein lysate was added into an 8% SDS-PAGE Tris-glycine gel and run using the Bio-rad Mini-Protean system (Bio-Rad, Hercules, CA). The gel was transferred onto a 0.2 mm nitrocellulose membrane using Bio-rad Trans-Blot Turbo Transfer system (Bio-Rad, Hercules, CA). An antibody against the C-terminal end of TDP-43 (10782–2-AP, ProteinTech, Rosemont, IL) was used for immunoblotting. The immunoblots were imaged using the LI-COR Odyssey Infrared Imaging system (Li-COR, Lincoln, NE).
7
Autophagy Protein Expression Analysis
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Western blot analysis was used for the evaluation of autophagy proteins. Total protein lysates (40 µg/lane) were separated by electrophoresis on SDS PAGE gels and then transferred to PVDF membranes, using the Bio-Rad Miniprotean system (Bio-Rad). Blots were blocked with StartingBlock Buffer (Thermo Fischer Scientific) and then incubated with primary antibodies against Beclin 1, ATG4b, ATG5, ATG9a, ATG16L1, LC3B (Abcam ab228525), IL-6 (Abcam), and GAPDH (Santa Cruz; sc-166545). Detection was done using Supersignal West Femto-Chemiluminiscent substrate (Thermo Fisher Scientific, Rockford, IL, USA). Image acquisition was done using ChemiDoc System (Bio-Rad) while quantification was done using Image Lab analysis software (Bio-Rad, Hercules, CA, USA). GAPDH was used as a protein loading control. All investigated proteins were normalized on GAPDH after which DOX-treated samples were represented as a fold change of their respective untreated controls.
8
Western Blot Analysis of Muscle Protein
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For western blotting analysis, the muscle biopsy from IV to 11 was homogenized in the Laemmli sample buffer and heated at 95°C for 5 minutes. The soluble fraction after centrifugation (13,000 × g, 10 minutes) was collected and used for SDS-PAGE using 4%–20% TGX gradient gels and Bio-Rad Mini-PROTEAN system (Bio-Rad Laboratories, CA). After separation, the proteins were transferred onto PVDF membranes using the Bio-Rad TransBlot Turbo device. The membranes were incubated with anti-HNRNPA1 (Abcam ab5832) antibody at 1:10,000, diluted in 5% nonfat milk powder/TBST, overnight at +8°C. After HRP-conjugated secondary antibody incubation, ECL reaction (SuperSignal West Femto, Thermo Fisher Scientific) and Bio-Rad ChemiDoc reader were used for detection of bands. The gels were recovered after transfer and stained with Coomassie, and the myosin heavy chain bands were used as loading control.
9
Immunoblot Analysis of Apoptosis Markers
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Lysates (20 µg protein/lane) underwent electrophoresis on SDS PAGE gels, followed by transfer to polyvinylidene difluoride membranes using the Biorad Miniprotean system (Bio-Rad Laboratories, Hercules, CA, USA). The blots were blocked and then incubated with antibodies against p53, BCL-2, BAX, COX2, NFκB, pNFκB, and NRF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), as well as caspase-3 and caspase-9 (Antibodies Online, Atlanta, GA, USA). Subsequently, the blots were washed and exposed to corresponding secondary peroxidase-linked antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein detection was performed using Supersignal West FemtoChemiluminiscent substrate (Thermo Fisher Scientific, Rockford, IL, USA), and analysis was conducted using a Gel Doc Imaging system equipped with a XRS camera and Quantity One® 1-D analysis software 4.6 (Bio-Rad Laboratories, Hercules, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA) served as a protein-loading control.
10
Evaluation of Inflammatory Mediators
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Inflammation was assessed by the measurement of IL-31, IL-33, IL-6, TNF-α, and STAT3 levels using ELISA immunoassay kits from R&D Systems Inc. (Minneapolis, MN, USA). In addition, the expressions of transcription factor NFκB and its phosphorylated form (pNFκB) were evaluated by western blot analysis. For western blot, a 20 μg protein/lane was separated by electrophoresis on SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes (Bio-Rad Mini-PROTEAN System from Bio-Rad) as previously described [46 (link)]. Blots were blocked and then incubated with antibodies against NFκB, pNFκB p65 (Ser536) (93H1), and GAPDH and then further washed and incubated with corresponding secondary peroxidase-linked antibodies (Santa Cruz Biotechnology Inc.). The proteins were detected using the SuperSignal West Femto chemiluminescent substrate (Thermo Fisher Scientific, Rockford, IL, USA) and were then quantified using Quantity One Analysis Software (Bio-Rad).
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