All bacteriological grade growth medium and reagents were purchased from Himedia. Analytical reagents for enzyme assay, TLC, SDS-PAGE, buffers, B. amyloliquefaciens alpha amylase, dialysis membrane, and TLC Plates were purchased from Sigma-Aldrich. The starch quantification kit was purchased from Megazyme. The dry biomass of Chlorella vulgaris was obtained from the Algae Research Laboratory, Department of Biotechnology, Kathmandu University. All statistical analyses were done in Minitab 19.0.
Sds page
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SDS-PAGE is a widely used analytical technique in biochemistry and molecular biology laboratories. It separates proteins based on their molecular weight by using sodium dodecyl sulfate (SDS) to denature and apply a uniform negative charge to the proteins, which are then subjected to electrophoresis through a polyacrylamide gel matrix.
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Lab products found in correlation
Pvdf membrane, by Merck Group (17 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (11 mentions)
Polyvinylidene difluoride membrane, by Merck Group (6 mentions)
Ripa buffer, by Merck Group (5 mentions)
Protein assay, by Bio-Rad (4 mentions)
Skim milk, by BD (4 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
Ab8245, by Abcam (4 mentions)
Bca protein assay kit, by Merck Group (3 mentions)
Ecl substrate, by Merck Group (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Gapdh, by Santa Cruz Biotechnology (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (3 mentions)
Ab37168, by Abcam (3 mentions)
Gapdh, by Abcam (3 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (3 mentions)
Runx2, by Merck Group (2 mentions)
Hsp90, by Santa Cruz Biotechnology (2 mentions)
84 protocols using sds page
1
Enzymatic Starch Hydrolysis by Bacillus amyloliquefaciens
2
SDS-PAGE and Proteomic Analysis
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SDS-PAGE was performed with 4%–20% precast gels in Tris-based buffer system from SERVA Electrophoresis (#43289.01) according to the manufacturer’s instructions. PageBlue Protein Staining Solution (Fermentas, #R0571) was used for coomassie-staining the gels, images were taken with iPhone 6s (Apple) and the contrast was adjusted using ImageJ (version 1.52d). Global identification and quantification of MSU- and zymosan-binding proteins in the presence of different donor sera was done as previously described (25 (link)). In brief, eluted proteins were reduced with DTT, alkylated with acrylamide, and separated using SDS-PAGE (4%–20%, Sigma-Aldrich). Whole lanes were cut into three individual slices and proteins therein were in-gel digested with trypsin. Generated peptides were analyzed using an LC-MS system consisting of an Orbitrap Velos mass spectrometer coupled to an Ultimate 3000 RSLC nanoflow system (Thermo Fisher Scientific). Raw data were analyzed with the Andromeda search engine implemented in MaxQuant software (version 1.5.3.30; www.maxquant.org ). Proteins were identified based on a false discovery rate (FDR) of less than 0.01 on protein and peptide level.
3
Immunoblotting Analysis of Hypothalamus, Pituitary, and Ovary
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After weighing the hypothalamus, pituitary gland and ovary, RIPA lysate was added at the ratio of weight: lysate volume equal to 1:9. The tissue homogenate was centrifuged three times at 15,000 g for 10 s. After incubation on ice for 20 min, the supernatant was centrifuged at 4 °C and 13,000 g. Then, the supernatant was transferred to PVDF membrane by SDS-PAGE (Sigma), which was subsequently taken out and soaked in 3% BSA-TBST, and then sealed for 30 min by shaking at room temperature. After the blocking solution was discarded, IFN-γ antibody was added at a dilution ratio 1:10,000, and the solution was incubated for 10 min at room temperature and then overnight at 4 °C. The membrane was washed five times with TBST for 3 min each time, and incubated 40 min at room temperature with the secondary anti mouse IgG (H + L) HRP at the dilution ratio of 1:10,000. Then wash the membrane by TBST 6 times, for 3 min each time. After ECL was added to the PVDF membrane for 3–5 min, the membrane was exposed for 10 s to 5 min (the exposure time was inversely proportional to the light intensity). Finally, developed for 2 min, and performed fixation. The gray values of protein bands were quantified by ImageJ software. Quantification of related proteins was normalized to β-actin levels.
4
SDS-PAGE Reagents and Chemicals
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Chemicals and reagents required for SDS-PAGE (Richmond, CA) were procured from Sigma Aldrich (St. Louis, MO, USA). Quercetin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), pancreatic α-amylase (VI-B ≥ 5 units/mg of solid), and yeast β-glucosidase (type I, lyophilized powder ≥ 10 units/mg of protein), ascorbic acid, H2O2 and rest of the chemicals were procured from Sigma Aldrich (St. Louis, MO, USA). All the chemicals used in the study were of analytical grade and were used without any further purification.
5
Exosomal Protein Marker Detection by Western Blot
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Exosomal protein makers were determined using western blot. Fifteen micrograms of EVs protein were resolved with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE, Sigma‐Aldrich) and blotted to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Bedford, MA, USA). The blotted membrane was blocked with 5% skim milk (Sigma‐Aldrich) for 1 h at room temperature followed by staining with anti‐CD81 (1:250, cat. 10630D, Invitrogen, La Jolla, CA, USA) and antiflotillin‐1 (1:2000, cat. F1180, Sigma‐Aldrich) at 4 °C overnight. The membrane was washed with buffer several times to remove primary antibodies before staining with horseradish peroxidase (HRP)‐conjugated secondary antibody for 1 h at room temperature. The chemiluminescent signal was developed using ECL Prime western blotting detection reagent (GE Healthcare, Chicago, IL, USA) and visualized by chemiluminescent detection instrument (Bio‐Rad, Hercules, CA, USA).
6
Western Blot Analysis of ALS-associated Proteins
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Ctrl (n = 5), C9orf72 (n = 3), FUS (n = 1), TARDBP (n = 2), and uvALS (n = 3) fibroblasts were lysed on plates in 2xLaemmli buffer and the lysates were boiled at 100 °C for 5 min. Spinal cords, sciatic nerves, and gastrocnemius muscles of n = 4 animals per group were dissected [1 (link)] and lysed in homogenization buffer (50 mM Tris HCl pH 7.4, 250 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 1% Triton X-100, 0.25% Na-deoxycholate, 0.1% SDS, protease inhibitor cocktail from Sigma-Aldrich). After 2 × 10″ sonication cycles, samples were incubated on ice and then centrifuged at 15,000×g for 20′ at 4 °C. Supernatants were then quantified with Bradford protein assay (Bio-Rad) and resuspended in Laemmli Buffer before SDS-PAGE (Sigma-Aldrich). Proteins were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes, followed by incubation with 5% skimmed milk for 1 h and with primary antibodies at 4 °C overnight. HRP-conjugated secondary antibodies (1:2,500, Jackson ImmunoResearch) were applied at RT for 1 h. ECL solution (Roche) was used for chemiluminescent detection. GAPDH was used as a control for equal loading. Following densitometry-based quantification and analysis using ImageJ software, the relative density of each identified protein was calculated.
7
Western Blot Analysis of Signaling Pathways
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Protein samples (20 µg each) were separated using 10 or 7.5% SDS-PAGE (Sigma-Aldrich, St. Louis, MO, USA) and electrotransferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA) according to a previously described method (16 ). Following blocking with 3% bovine serum albumin for 3 h, the membranes were probed with the following primary antibodies: phosphorylated (p)-ERKl/2 rabbit anti-mouse polyclonal antibody (cat. no. SC7383), p-P38MAPK monoclonal antibody (cat. no. bs-547612), p-JNK (cat. no. elr-0011876) and rabbit polyclonal anti-β-actin (cat. no. 4970P) (all 1:1,000 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA). Furthermore, Bcl-2 rabbit polyclonal anti-mouse antibody and Bcl-2-associated X protein (Bax) polyclonal rabbit anti-mouse antibody were used (both from Wuhan Boster Biological Engineering Co., Ltd., Wuhan, China). Membrane-bound antibodies were further detected using alkaline phosphatase-conjugated goat anti-mouse or rabbit immunoglobulin (Ig) G (1:10,000 dilution, Sigma-Aldrich). The immunoreactivity was assessed using a NBT/BCIP assay kit (Kexing Biological Technology Co., Ltd., Shanghai, China). The band densities on the membrane were measured using an image analyzer (Lab Works Software version 17.0; UVP Inc., Upland, CA, USA) and normalized to the internal control.
8
Quantifying ADAMTS4 and Aggrecan Levels
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To verify the impacts of AAV6-ADAMTS4 on the protein expression levels of ADAMTS4 and aggrecan western blot was applied. 1 x 105 NP cells were seeded, transduced, cultured on the scaffold and harvested on day 8 and 48 as described before. For total protein isolation harvested cells were lysed for 20 min by using ice cold radio-immunoprecipitation assay (RIPA) buffer (Sigma-Aldrich), which was supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). Samples were centrifuged for 10 min (14000 × g, 4°C) and supernatants were used for western blot analysis. Total protein concentration in samples and controls was determined by BCA Protein Assay Kit (Thermo Scientific). Equal amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Sigma-Aldrich) and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore). Anti-ADAMTA4 antibody (SAB1411668, Sigma-Aldrich) and anti-aggrecan antibody (SAB4500662, Sigma-Aldrich) were used as primary antibodies. Interactions between antigens and primary antibodies were detected on the membrane by using horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (A0545, Sigma-Aldrich) and Amersham ECL Western Blotting Detection kit (GE Healthcare Life Sciences). Image J software was used to quantify protein bands.
9
Western Blot Protein Analysis Protocol
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Proteins of from the previously mentioned cells or BMMs lysed through RIPA lysis buffer (Thermo Fisher Scientific, USA) were isolated by Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Sigma-Aldrich), which were transferred to Polyvinylidene fluoride (PVDF) membranes. The membranes blocked with 5% skim milk for 2 h at room temperature, were incubated with primary antibodies overnight at 4°C. And then the membranes were washed with Tris-buffered saline with Tween (TBST) buffer. Secondary antibody was utilized to incubate the membranes at room temperature for 1 h. The results were visualized by Pierce ECL Western Blotting Substrate kit for enhanced chemiluminescence (ECL). Antibodies are listed in Table S3.
10
Protein Expression Analysis of MSCs
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MSCs were lysed in passive lysis buffer (Promega, Madison, WI, USA). Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and 30 mg of protein was analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Sigma). Transferred membranes were blocked with 5% skim milk (BD, Sparks, MD, USA), and incubated for 10 h with antibodies against NRF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated-NRF2 (Abcam, Cambridge, UK), HDAC1 (Santa Cruz Biotechnology), HSP90 (Santa Cruz Biotechnology), LAMIN-B (Santa Cruz Biotechnology), LDH (Santa Cruz Biotechnology), RUNX2 (EMD Millipore, San Diego, CA, USA), SIRT1 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), and HIC1 (Santa Cruz Biotechnology). Membranes were further probed with an antibody against β-ACTIN (Santa Cruz Biotechnology, Dallas, TX, USA) that served as a loading control.
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