The specific method of western blot was consistent with our previous research [31 (link)]. Cell lysates were prepared using RIPA lysis buffer (Bimake, Houston, Texas, USA), and the proteins in cell lysates were separated using 10% SDS–PAGE and transferred into PVDF membranes (Millipore, Billerica, MA, USA). After being blocked with skimmed dry milk, PVDF membranes were incubated overnight with primary antibodies of CRABP2 (Proteintech, Chicago, Illinois, USA) and β-Actin (Santa Cruz, Dallas, Texas, USA). Later, Immobilon Western chemiluminescent reagents (Millipore, billerica, MA, USA) were used to detect proteins on the membranes.
Ripa lysis buffer
Manufactured by Selleck Chemicals
Sourced in United States
RIPA lysis buffer is a reagent used to extract and solubilize proteins from cells and tissues for analysis. It is a solution containing detergents, salts, and buffers that disrupt cell membranes and protein-protein interactions, allowing for the extraction of a wide range of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Immobilon western chemiluminescent reagent, by Merck Group (1 mentions)
Pvdf membrane, by Proteintech (1 mentions)
Crabp2, by Proteintech (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor, by Selleck Chemicals (1 mentions)
Beyoecl plus, by Beyotime (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Imagequant las 4000 system, by GE Healthcare (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitors and phosphatase inhibitors, by Selleck Chemicals (1 mentions)
Anti hnf 4α, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
4 protocols using ripa lysis buffer
1
Western Blot Analysis of CRABP2
2
Analyzing CXCR3 Expression in Mouse Spinal Cord
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The cervical spinal cord of mice was homogenized in the ice-cold RIPA lysis buffer (Bimake, China) containing protease inhibitors (Bimake, China) and phosphatase inhibitors (Roche, Germany) (Xu et al., 2021 (link)), lysed for 30 min, and then centrifuged at 12,000 rpm 4°C for 10 min. Next, the supernatant was mixed with 5× SDS loading buffer and incubated at 95°C for 10 min. Then, the boiled protein lysate was separated by 10% SDS-PAGE and transferred to 0.22 μm PVDF membranes. After blocking with 5% BSA for 2 h at room temperature (RT), the membranes were incubated with primary antibodies at 4°C overnight, washed in TBST, and incubated with appropriate HRP-conjugated secondary antibodies for 2 h at RT. The protein bands were visualized by using BeyoECL Plus (P0018, Beyotime, China), and a densitometry analysis was performed on an imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China) (Liu S. et al., 2019 (link)). The primary antibodies used in this study are anti-CXCR3 (26756-1-AP, Proteintech, 1:1,000) and anti-GAPDH (#6176106, CST, 1:10,000).
3
Western Blot and Co-immunoprecipitation Assay
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The cells were lysed with ice-cold RIPA lysis buffer (Bimake, Shanghai, China). A BCA Protein Assay Kit (Pierce, CA) was used to determine the protein concentration. About 10–30 μg of total protein was resolved by 8%–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore, Billerica, MA). The membranes were blocked with 5% non-fat dried milk in 1× TBST buffer and incubated at 4°C for at least 16 hours with the indicated primary antibodies. Secondary antibodies conjugated with HRP anti-rabbit (1:2,000, Proteintech) or anti-mouse (1:2,000, Proteintech) were used. The membranes were incubated with Immobilon Western Chemiluminescent HRP Substrate (Thermo Fisher Scientific, Waltham, MA). The chemiluminescent signal was detected by the ImageQuant LAS 4000 system (GE Healthcare, Chicago, IL). The band intensities were quantified using ImageJ software and normalized to the expression of β-actin or GAPDH (loading control) and expressed as fold-change relative to control group.
For co-immunoprecipitation, cell lysate was incubated with a chosen antibody overnight at 4°C followed by incubation with magnetic beads (Pierce) for 1 hour at RT. Beads were washed twice with lysis/wash buffer and once with purified water. The antigen/antibody complex was eluted. Then, samples were subjected to SDS-PAGE for separation and detection.
For co-immunoprecipitation, cell lysate was incubated with a chosen antibody overnight at 4°C followed by incubation with magnetic beads (Pierce) for 1 hour at RT. Beads were washed twice with lysis/wash buffer and once with purified water. The antigen/antibody complex was eluted. Then, samples were subjected to SDS-PAGE for separation and detection.
4
Protein Expression Analysis in Liver
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Liver tissues were lysed in RIPA lysis buffer with protease inhibitors and phosphatase inhibitors (Bimake, Houston, USA) for total protein according to the manufacturer’s instructions (Shenergy Biocolor Bioscience & technology CO., Shanghai, China). Protein concentration was measured using BCA Protein Quantitative Assay Kit (Shenergy Biocolor Bioscience & technology CO., Shanghai, China). The target proteins were blotted with the following antibodies: anti-HNF4α (41770, Invitrogen, USA), anti-SIRT1 (sc-15404, Santa Cruz Biotechnolosy, USA), anti-PPARα (MAB3890, Merck KGaA, Germany), anti-GAPDH (60004–1, Proteintech, China). The relative target protein levels were quantified by densitometry normalized to GAPDH on the same membrane. The samples in one group from three different mice and three independent experiments were performed.
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