Lysates for SDS-PAGE analysis were prepared in lithium dodecylsulphate sample buffer (Life Technologies) and 25 mM TCEP. Samples were heated to 65°C for 5 min and then loaded onto a NuPage BisTris 4–12% gradient gel (Life Technologies), in either MOPS, or MES buffer. Proteins were electrophoresed and then wet transferred to nitrocellulose membranes at 35 V for 1.5 hr. Membranes were then blocked in 5% BSA in immunoblot wash buffer (TBS +0.1% Tween-20) for 1 hr at room temperature. Membranes were then probed with primary antibody overnight at 4°C, washed and then re-probed with LiCor dye-conjugated secondary antibodies (either IRDye-688 or IRDye-800). Primary antibodies for cell cycle immunoblot analysis were obtained from Cell Signaling Technology (cyclin B1, cyclin A, cyclin E, CDT1). Bands were visualised using the Odyssey CLx scanner (LiCor Biosciences).
Lithium dodecylsulphate sample buffer
Manufactured by Thermo Fisher Scientific
Lithium dodecylsulphate sample buffer is a laboratory reagent used to prepare samples for analysis. It is a detergent-based buffer that helps to solubilize and denature proteins, making them suitable for further processing and analysis.
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Lab products found in correlation
Nupage bistris 4 12 gradient gel, by Thermo Fisher Scientific (1 mentions)
Odyssey clx scanner, by LI COR (1 mentions)
Sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Dialysis tubing, by Merck Group (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Nupage antioxidant, by Thermo Fisher Scientific (1 mentions)
Mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Simplyblue safe stain solution, by Thermo Fisher Scientific (1 mentions)
Iblot gel transfer stack, by Thermo Fisher Scientific (1 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (1 mentions)
2 protocols using lithium dodecylsulphate sample buffer
1
Cell Cycle Protein Immunoblot Analysis
2
Detailed Hair Keratin Extraction Protocol
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Hydrogen peroxide (H2O2), sodium sulphide (Na2S), N-Ethylmaleimide (NEM) and dialysis tubings (molecular weight cut-off 12,400 Da) were purchased from Sigma Aldrich. Dulbecco's modified Eagle's medium (DMEM), L-glutamine, sodium pyruvate, 0.25% trypsin, PicoGreen DNA quantification kit, antibody-antimycotic mixture, secondary goat anti-mouse horseradish-peroxidase conjugated (HRP) antibody, CM-H2DCFDA, SimplyBlue SafeStain solution, precast 4-12% Bis-TRIS NuPAGE gels, lithium dodecyl sulphate sample buffer, sample reducing agent, MOPS SDS running buffer, NuPAGE antioxidant, and iBlot gel transfer stacks were purchased from Life Technologies. Primary mouse polyclonal antibody against total human hair keratins (clone AE13, #ab16113) was obtained from Abcam. The Super Signal West Pico chemiluminescent substrate was purchased from Pierce.
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