Tumor tissues obtained from mice in each experimental group were homogenized in cold lysis buffer with 1 mM phenylmethanesulfonyl fluoride and subsequently lysed for 60 minutes on ice. Protein concentrations were determined via Coomassie Blue Fast Staining (Beyotime Institute of Biotechnology, Shanghai, China). Protein samples (50 μg) from each experimental group were separated via 10% SDS-PAGE and subsequently transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Following this, membranes were blocked using Tris-buffered saline containing Tween 20 and 5% skimmed milk for 3 hours and subsequently incubated with the survivin antibodies (1:750) overnight at 4°C. GAPDH antibodies (1:15,000) were used as an endogenous reference. Following this, membranes were incubated with secondary antibodies (1:4,000) at room temperature for 1 hour. The blots were then visualized using Immobilon™ Western Chemiluminescent Horseradish Peroxidase Substrate (EMD Millipore) and the Tanon-4500 Gel Imaging System and subsequently quantified using GIS ID Analysis Software v4.1.5 (Tanon Science & Technology Co., Ltd., Shanghai, China).
Coomassie blue fast staining
Manufactured by Beyotime
Sourced in China
Coomassie Blue Fast Staining is a reagent used for the rapid staining of proteins in gels. It is a simple and effective method for visualizing and quantifying proteins separated by gel electrophoresis.
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2 protocols using coomassie blue fast staining
1
Western Blot Analysis of Survivin in Tumor Tissues
2
Protein Extraction and Immunoprecipitation
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Proteins were extracted by Cell Lysis Buffer for Western & IP (Beyotime) supplemented with phosphatase and proteinase inhibitor. Primary antibodies against Anti-MYC-Tag mAb (DIA-AN, #2097), FLAG-Tag mAb (DIA-AN, #2064), LKB1 (27D10) Rabbit mAb (CST, #3050), Rb (D20) Rabbit mAb (CST, #9313) and p65 (CST, #8242) were added. The mixtures were incubated at 4 °C with gentle shaking overnight. Protein A/G agarose beads were added and allowed to incubate for another 2 h. The proteins were precipitated through centrifugation and dissolved in SDS sample buffer. Samples were then fractionated by 10% SDS-PAGE gel. Coomassie Blue Fast Staining (Beyotime, #P0017) was performed according to the manufacturer’s protocols.
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