Liver tissues were homogenized in 25 mM HEPES buffer with 5 mM EDTA (pH 7.4), 0.1% CHAPS, and protease inhibitors, using a bead homogenizer (Thermo Fisher Scientific, Waltham, MA). Protein concentration in the homogenates was measured using the bicinchoninic acid (BCA) assay, and equal amounts of protein were loaded in each lane of a 4-20% Tris-glycine gel. After electrophoresis, proteins were transferred to PVDF membranes with 0.45 μm pores and blocked with 5% milk in Tris-buffered saline with 0.1% Tween 80. After incubation with the appropriate antibodies, protein bands were visualized using the Odyssey Imaging System (LiCor Biosciences, Lincoln, NE). All primary antibodies were used at a 1:1,000 dilution. Primary antibodies were purchased from Cell Signaling Technology (Danvers, MA): phosphorylated GSK3β (Cat. No. 9323), GSK3β (Cat. No. 9315), PCNA (Cat. No. 2586), and β-actin (Cat. No. 4967). Secondary antibodies were purchased from Li-Cor (Lincoln, NE): IRDye 680 goat anti-mouse IgG (Cat. No. 926-68070) and IRDye 800CW goat anti-rabbit IgG (Cat. No. 926-32211) were used at a 1:10,000 dilution.
Bead homogenizer
Manufactured by Thermo Fisher Scientific
The Bead homogenizer is a versatile laboratory equipment designed for efficient sample disruption and homogenization. It utilizes high-speed agitation with beads to physically break down and mix a wide range of sample materials, including tissues, cells, and other solid or semi-solid substances. The Bead homogenizer ensures thorough and consistent sample preparation for downstream analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 680 goat anti mouse igg, by LI COR (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Phosphorylated gsk3β, by Cell Signaling Technology (1 mentions)
Microcentrifuge tube, by Eppendorf (1 mentions)
Purelink microbiome dna purification kit, by Thermo Fisher Scientific (1 mentions)
Elution buffer, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
2 protocols using bead homogenizer
1
Western Blot Analysis of Phosphorylated GSK3β
2
Fecal DNA Extraction Protocol
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Mice were placed in an empty autoclaved glass beaker and one fresh fecal pellet was transferred to a sterile 2 ml round-bottom tube (Nalgene, Rochester, NY) on dry ice and stored at − 80 °C until further analysis. For DNA extraction, lysis buffer was adjusted by fecal weight and up to 800 μl was added per bead-containing tube following the manufacturer’s instructions using Purelink Microbiome DNA Purification Kit (Invitrogen, Carlsbad, CA, USA). Samples were homogenized (30 s) in a bead homogenizer (Thermo Fisher, Waltham, MA) and centrifuged at 14000 g (5 min), and the supernatants were collected in a 1.5 mL microcentrifuge tube (Eppendorf, Hauppauge, NY). Samples were transferred to a spin column for purification and eluted in 100 μl elution buffer (Invitrogen by Thermo Fisher). DNA concentrations were determined by spectrophotometry (Nanodrop, Wilmington, DL). The purified DNA was used for bacterial 16S rRNA qPCR to monitor potential contamination. For bacterial 16S rRNA gene sequencing, DNA of feces from GF mice was extracted using QIAamp DNA Stool Mini kit following the manufacturer’s instructions (Qiagen Ltd., Strasse, Germany) for quality control.
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