Dca protein assay kit

HEK293T cells were transiently transfected and inhibitor treated as indicated. OCI-AML2 and MV-4-11 cells were plated in 6-well culture plates at 1.0 × 10⁶ cells/well and treated with VbP as indicated. Supernatants were analyzed for LDH levels using the Pierce LDH Cytotoxicity Assay Kit (Life Technologies). LDH levels were quantified relative to a lysis control where cells were lysed in 80 μL of a 9% Triton X-100 solution. For FLAG immunoprecipitation, cell pellets were lysed in Tris-Buffered Saline (TBS) with 0.5% NP-40 on ice for 20 min and centrifuged at 20000 x g for 10 min. The soluble fraction was retained, and protein concentrations were normalized to 1 mg/mL using the DCA Protein Assay kit (Bio-Rad). Lysates were incubated with 40 μL of anti-FLAG-M2 agarose resin (Sigma) overnight at 4°C. After washing 3 × 1 mL with PBS, bound proteins were eluted by incubating resin with 100 μL of PBS containing 150 ng/ μL 3 × -FLAG peptide for 1 h at 4°C. An equal volume of 2 × sample loading dye was added to the eluate and incubated at 95°C for 10 min. For immunoblotting, cells were washed 2 × in PBS (pH = 7.4), resuspended in PBS, and lysed by sonication. Protein concentrations were determined and normalized using the DCA Protein Assay kit (Bio-Rad). The samples were separated by SDS-PAGE, immunoblotted, and visualized using the Odyssey Imaging System (Li-Cor).

Cells were washed 2 × in PBS (pH = 7.4), resuspended in PBS, and lysed by sonication. Protein concentrations were determined and normalized using the DCA Protein Assay kit (Bio-Rad). To prepare supernatant western blotting samples of N/TERT-1 keratinocytes, spent media of each treatment were combined, centrifuged at 400 g for 3 min, and the supernatant was precipitated by addition of four sample volumes of acetone at −20°C overnight followed by centrifugation at 3000 g for 30 min at 4°C and decanting. Protein pellets were then suspended in 1X PBS and combined 1:1 with 2X sample loading buffer before heating to 97°C for 10 min.
Samples were run on NuPAGE 4 to 12%, Bis-Tris, 1.0 mM, Midi Protein Gel (Invitrogen) for 45–60 min at 175 V. Gels were transferred to nitrocellulose with the Trans-Blot Turbo Transfer System (BIO-RAD). Membranes were blocked with Intercept (TBS) Blocking Buffer (LI-COR) for 30 min at ambient temperature, before incubating with primary antibody overnight at 4°C. Blots were washed 3 times with TBST buffer before incubating with secondary antibody for 60 min at ambient temperature. Blots were washed 3 times, rinsed with water, and imaged via Odyssey CLx (LI-COR).