Each uterine sample was prepared from a single uterine horn of one mouse. The uterine horn was cut into small pieces and lysed in a radioimmunoprecipitation assay buffer: 10 mM Tris (pH 7.2; HanLAB), 150 mM NaCl (Fisher Scientific), 0.1% Triton X-100 (Sigma-Aldrich), 5 mM ethylenediaminetetraacetic acid (EDTA; HanLAB), 1% sodium dodecyl sulfate (Bio-Rad Laboratories Inc.), 1 mM dithiothreitol (Sigma-Aldrich), 1 mM phenylmethylsulfonyl fluoride (PMSF; MP Biomedicals), and 1X protease inhibitor (Roche). We used a Kinematica Polytron homogenizer (Kinematica AG), and centrifuged the samples at 15,928 × g for 20 minutes at 4 °C. Protein concentrations were determined by bicinchoninic acid protein assay (Thermo Fisher Scientific). Lysates (10 μg) were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred onto nitrocellulose membranes (Bio-Rad). After Western blotting, the chemiluminescence signals were detected using a West Femto kit (SuperSignal, Thermo Fisher Scientific). The intensity of the signal was normalized against the β-tubulin signal. The data were presented as mean±standard error of the mean. The primary antibodies used were rabbit polyclonal anti-ATG9A antibody (26276-1-AP; Proteintech Genomics), rabbit polyclonal anti-ATG9B (ab117591; Abcam), and rabbit polyclonal anti-β-tubulin antibody (ab6046; Abcam).
Sodium dodecyl sulfate (sds)
Manufactured by Bio-Rad
Sourced in United States, United Kingdom, Germany, Italy, France
SDS (Sodium Dodecyl Sulfate) is a laboratory reagent commonly used in biochemical and molecular biology applications. Its core function is to denature proteins by disrupting non-covalent interactions, which aids in the separation and analysis of protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Acrylamide, by Bio-Rad (15 mentions)
Ammonium persulfate, by Bio-Rad (13 mentions)
Dithiothreitol (dtt), by Merck Group (13 mentions)
Urea, by Bio-Rad (11 mentions)
Ammonium bicarbonate, by Merck Group (11 mentions)
Triton x 100, by Merck Group (11 mentions)
Trypsin, by Promega (10 mentions)
Formic acid, by Merck Group (10 mentions)
Iodoacetamide, by Merck Group (10 mentions)
Glycine, by Bio-Rad (10 mentions)
Glycerol, by Merck Group (10 mentions)
Acetonitrile, by Merck Group (8 mentions)
Methanol, by Merck Group (8 mentions)
Bromophenol blue, by Bio-Rad (8 mentions)
β mercaptoethanol, by Merck Group (8 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Sodium chloride (nacl), by Merck Group (8 mentions)
Bovine serum albumin (bsa), by Merck Group (7 mentions)
Sodium carbonate, by Merck Group (7 mentions)
Glycine, by Merck Group (7 mentions)
151 protocols using sodium dodecyl sulfate (sds)
1
Immunoblotting Analysis of Uterine Samples
2
Cell Lysis and Protein Extraction
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Culture medium from cells growing on dishes was either removed or non-adherent cells/cellular material recovered from the medium by centrifugation (400 × g at room temperature (RT) for 3 min (min)). Cells were washed with PBS and then lysed for 5 min with ice-cold RIPA lysis buffer (20 mM Tris-HCl (pH 7.4), 137 mM NaCl, 1 mM EGTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS (Bio-Rad, Watford, UK), 1% (w/v) sodium deoxycholate, 10% (v/v) glycerol, 1.5 mM MgCl2, 50 mM NaF, 1 mM Na3VO4, 5 μg mL−1 aprotinin, 10 μg mL−1 leupeptin, 1 mM PMSF, 0.025 U mL−1 Benzonase). Lysates were detached and collected using a cell scraper and transferred to pre-chilled tubes. Protein concentration was determined by BCA protein assay (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, Loughborough, UK) and absorbance measured at 562 nm using a PHERAstar FS plate reader (BMG Labtech, Aylesbury, UK). Samples were prepared for SDS-PAGE by boiling for 5 min in 1 × Laemmli sample buffer (50 mM Tris-HCl (pH 6.8), 2% (w/v) SDS (Bio-Rad, Watford, UK), 10% (v/v) glycerol, 1% (v/v) β-mercaptoethanol, 0.01% (w/v) bromophenol blue). Unless otherwise indicated, reagents were from Sigma-Aldrich, Dorset, UK.
3
Homemade Laemmli Gel Electrophoresis
4
Protein Extraction from PDX Tumor Tissues
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Colorectal and melanoma PDX tumour tissue was from the internal AstraZeneca PDX library. Snap frozen tumour fragments were homogenised in ice-cold RIPA lysis buffer (20 mM Tris-HCl (pH 7.4), 137 mM NaCl, 1 mM EGTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS (Bio-Rad, Watford, UK), 1% (w/v) sodium deoxycholate, 10% (v/v) glycerol, 1.5 mM MgCl2, 50 mM NaF, 1 mM Na3VO4, 5 μg mL−1 aprotinin, 10 μg mL−1 leupeptin, 1 mM PMSF, 0.025 U mL−1 Benzonase) using gentleMACS M tubes and a gentleMACS Dissociator on setting Protein_0.1.01 (Miltenyi Biotec, Surrey, UK). Lysates were collected by centrifugation (400 × g, 4 °C, 1 min) and protein concentration determined by BCA protein assay (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, Loughborough, UK) and absorbance measured at 562 nm using a PHERAstar FS plate reader (BMG Labtech, Aylesbury, UK). Samples were prepared for SDS-PAGE by boiling for 5 min in 1 × Laemmli sample buffer (50 mM Tris-HCl (pH 6.8), 2% (w/v) SDS (Bio-Rad, Watford, UK), 10% (v/v) glycerol, 1% (v/v) β-mercaptoethanol, 0.01% (w/v) bromophenol blue). Unless otherwise indicated, reagents were from Sigma-Aldrich, Dorset, UK.
5
Forced Swim Assay for Drosophila Behavioral Phenotyping
6
Quantitative Milk Protein Analysis
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We obtained the materials from Sigma-Aldrich (Saint Louis, MO, USA): bovine BC, a-LA, b-LG (purity ≥ 98%) and IgG (≥95%) protein standards, human milk LF protein standard (>95%), 1,4-dithiothreitol (DTT), iodoacetamide, acetonitrile, formic acid, and thermolysin (Type X, E.C. No. 3.4.24.27). Tris base, ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), ammonium persulfate, N, N, N, N’-tetramethylethylenediamine (TEMED), Precision Plus ProteinTM standards, and colloidal Coomassie G-250 stain, all molecular biology grade, were acquired from Bio-Rad (Hercules, CA, USA). Acrylamide, N, N’-methylenebisAcrylamide, urea, 2-mercaptoethanol, glycine and bromophenol blue, and ultrapure Bioreagent grade, as well as isobutyl alcohol, glacial acetic acid, and sodium hydroxide, were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Thermo Fisher Scientific provided Invitrogen’s Qubit® Protein Assay kit. Zip Tips C18 were obtained from Millipore Sigma (Saint Louis, MO, USA). Deionized water, used in all experiments, was purified using a Milli-Q system from Millipore Merck (Darmstadt, Germany).
7
Stage-specific Parasite Extraction
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For the stage-specific pull-down analysis parasites were harvested at ring stage (approx. 12HPI), trophozoites (approx. 24HPI) and schizonts (approx. 36HPI) Parasites were liberated, and red blood cells lysed using 10 volumes of 0.1% (w/v) saponin (Sigma) in ice-cold 1 x PBS (1st Base) for 5 minutes. Parasite pellet was washed three times with 1 x PBS and the pellet was lysed (for most of assays except Pull-downs) with RIPA buffer (150 mM sodium chloride (Merck); 1% Triton X-100 (BioRad); 0.5% sodium deoxycholate (Sigma); 0.1% sodium dodecyl sulfate (BioRad); and 50mM Tris (BioRad) pH8.0) supplemented with 1% (v/v) protease inhibitor cocktail (Nacalai Tesque) and 1% (v/v) phosphatase inhibitor cocktail (Thermo Fisher). Samples were incubated for 30 min at 4°C with gentle agitation. For native pull-down assays RIPA buffer was substituted with Pierce IP Lysis Buffer (Thermo Fisher) with 1% (v/v) protease inhibitor cocktail (Nacalai Tesque) and 1% (v/v) phosphatase inhibitor cocktail (Thermo Fisher). Protein lysate debris was removed by centrifugation (15 min at 4°C) and protein concentration in the supernatant was quantified using Pierce BCA Protein Assay Kit (Thermo Scientific). Samples were frozen with liquid nitrogen and stored at -80°C.
8
Mass Spectrometry Sample Preparation
9
Collagen-based Cell Culture Protocol
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Rat tail type I collagen and protein marker (26634) were purchased from the Sigma Chemical Company (St. Louis, MO, USA), while sodium dodecyl sulfate (SDS), N, N, N, N-tetramethylethylenediamime (TEMED), and Coomassie Brilliant Blue R-250 were purchased from Bio-Rad Laboratories (Harkles, CA, USA). MC3T3-E1 cell lines (Cat No. CBP60946) were purchased from Cobioer (Nanjing, China). Other reagents used in the experimental process were of analytical-grade purity.
10
Reagents and Materials for Biochemical Analyses
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Bradford reagent, water CHROMASOLV®Plus for HPLC, bromophenol blue ACS reagent, ammonium bicarbonate BioUltra (≥99.5%) and acetonitrile anhydrous (99.8%) wereall acquired from Sigma-Aldrich (Milan, Italy). A 40% acrylamide/bis solution, ammonium persulfate, sodium dodecyl sulfate (SDS), trimethamine and glycine were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Boric acid and ethylenediaminetetraacetic acid (EDTA) were acquired from Carlo Erba (Milan, Italy). Glycerol 99.0–101.0% was purchased from Honeywell Research Chemicals (Seelze, Germany). Coomassie R-250 solution was purchased fromAmersham Pharmacia Biotech (Uppsala, Sweden). Agarose CSL-AG5 for gel electrophoresis was purchased from Cleaver Scientific (Rugby, UK). Gel red nucleic acid gel stain 10,000× in water was purchased from Biotium (Fremont, CA, USA). Tetramethylethylendiamine (TEMED) was purchased from Chem-Lab (Zedelgem, Belgium). GeneRuler DNA Ladders 10Kpb was purchased from Thermo Fisher Scientific Inc. (Swindon, UK). All other reagents were of analytical grade.
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