Novex nupage lds sample buffer

ApoE protein expression was detected in neuronal lysate and astrocyte and neuron conditioned media by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blot. Medium was collected and centrifuged at 10,000 × g for 10 min at 4°C before use. Neuronal lysate was washed in PBS, homogenized/lysed in a buffer containing 20 mM Tris, 150 mM KCl, 5 mM MgCl₂, and 1% NP40 with Halt^TM Protease inhibitor cocktail (Thermo Fisher Scientific, 78430) and Halt^TM Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, 78428), and centrifuged at 20,000 × g for 20 min at 4°C. The supernatant, which contained the intracellular proteins, was further used for Western blot. The media and lysate samples were mixed with Novex NuPage LDS sample buffer (Invitrogen, NP0007) and NuPage sample reducing agent (Invitrogen, NP0004), heated for 10 min at 70°C and loaded on a NuPAGE^TM 4–12% Bis-Tris protein gel (Invitrogen, NP0321). Secreted proteins in medium and intracellular proteins in lysate were separated by SDS PAGE. SeeBlue^® Plus2 pre-stained protein standard was used as a molecular weight marker.

Western blotting was performed with polyclonal peptide antibodies against either Ryp1, Ryp2, or Ryp3; antibodies were described previously by Beyhan and colleagues [2 (link)]. Following quantification of protein, 10 to 30 μg was resuspended in a total of 20 μL of urea lysis buffer and 6 μL Novex NuPAGE LDS Sample Buffer (Invitrogen, Carlsbad, CA). The samples were boiled and electrophoresed on a 10-well Novex NuPAGE 4% to 12% BT SDS-PAGE gel (Invitrogen, Carlsbad, CA) in MOPS running buffer at 150 V. The protein was then transferred to a nitrocellulose membrane at approximately 40 V for 2 hours. The membrane was incubated with blocking solution (1 g milk powder in 100 mL wash buffer [0.1% Tween-20 in PBS]) for an hour and then incubated in the primary antibody in wash buffer overnight at 4 °C. Primary antibody dilutions were: α-Ryp1 (1:10,000), α-Ryp2 (1:2,500), α-Ryp3 (1:5,000), α-GAPDH (1:1,000). The blot was washed and secondary antibody (for Ryp proteins: Goat α-rabbit HRP [GenScript, Piscataway, NJ] 1:1,000, for GAPDH: Goat α-mouse HRP [ThermoFisher, Weltham, MA] 1:1,000) was added to the blot for 1 hour at RT followed by another wash. Protein bands were detected using chemiluminescence according to the manufacturer’s instructions (SuperSignal West Pico kit; ThermoFisher, Weltham, MA).

Cells were seeded in 6-well plates 24 hours prior to transfection or drug treatment. Samples were harvested 48 hours after drug treatment and 72 hours after transfection. Cells were lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails. Running samples were prepared with 4X Novex NuPAGE LDS sample buffer (Life Technologies) and supplemented with 10% beta-mercaptoethanol. Twenty micrograms of protein were loaded per lane of 4–12% Bis-Tris NuPAGE gels (Life Technologies) and ran in MOPS buffer. Proteins were then transferred to PVDF-FL membrane (Millipore) in Tris-Glycine transfer buffer. Primary antibody incubation was carried out overnight at 4°C on a rocking platform. Secondary antibody incubation was carried out at room temperature the next day. Proteins were detected using the LI-COR Odyssey imaging system (LI-COR Biosciences). Image analysis and protein quantitation were performed with ImageJ (NCI).

Cells were lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails, and protein was quantified using BCA assay. Samples were prepared with 4X Novex NuPAGE LDS sample buffer (Life Technologies) and supplemented with 10% beta-mercaptoethanol. Twenty micrograms of protein was loaded per lane of 4%–12% Bis-Tris NuPAGE gels (Life Technologies) and run in MOPS buffer. Following gel electrophoresis, proteins were transferred to a PVDF-FL membrane (Millipore) in tris-glycine transfer buffer. Primary antibody incubation was carried out overnight at 4 °C on a rocking platform. Incubation with IRDye-conjugated secondary antibodies was carried out at room temperature the next day. Proteins were detected using the LI-COR Odyssey imaging system (LI-COR Biosciences). Image analysis and protein quantitation (i.e., band densitometry) was performed with ImageJ (NIH, Washington, DC, USA).