Sodium hydrosulfide (NaHS), chloroauric acid (HAuCl4), hemoglobin (Hb), hydrochloric acid (HCl) and acetone were bought from Innochem. Ethylene glycol (EG), silver nitrate (AgNO3) and 3,3’-Dioctadecyloxacarbocyanine perchlorate (DiO) were obtained from Aladdin. Poly(viny pyrrolidone) (PVP, Mw ≈ 55,000) was purchased from Sigma-Aldrich (St. Louis). Perfluorohexane (PFO) was bought from Energy Chemical. Silver trifluoroacetate (CF3COOAg) was obtained from Adamas. Indocyanine green (ICG) was purchased from ARK Pharm, Inc. Hoechst 33342, Cytosol Protein Extraction Kit and phenylmethanesulfonyl fluoride (PMSF) were purchased from Beyotime Biotechnology. All reagents for cell culture were bought from Gibco. The purified deionized water was prepared by the Milli-Q plus system (Millipore, USA).
Cytosol protein extraction kit
Manufactured by Beyotime
Sourced in China
The Cytosol Protein Extraction Kit is a laboratory tool designed to isolate cytosolic proteins from cellular samples. It provides a standardized method for the efficient extraction and purification of cytosolic proteins, which are essential for various downstream analyses and applications.
Automatically generated - may contain errors
Lab products found in correlation
Phenylmethanesulfonyl fluoride pmsf, by Beyotime (1 mentions)
Milli q plus system, by Merck Group (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Nuclear protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Na k atpase, by Santa Cruz Biotechnology (1 mentions)
Adi kas pk017 f, by Enzo Life Sciences (1 mentions)
Bca method, by Applygen (1 mentions)
5 protocols using cytosol protein extraction kit
1
Synthesis and Characterization of Nanomaterials
2
Protein Extraction and Western Blot Analysis
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A membrane and Cytosol Protein Extraction Kit (Beyotime, #P0033) and Enhanced BCA Protein Assay Kit (Beyotime, # P0010S) were used for the extraction and concentration determination of total protein. Subsequently, the proteins were separated by SDS-PAGE and transferred to PVDF membrane (Aspen, China, #AS1021). Subsequently, the membranes were probed overnight at 4°C with primary antibodies to β-actin (1:1000), DUSP10 (1:1000), LC3-I/II (1:500), Atg5 (1:2000), Atg7 (1:1000), Beclin1 (1:2000), and p-Erk1/2 (1:1000). Then, the membranes were incubated with secondary antibody (HRP-labeled goat anti-rabbit IgG, 1:2000) for 30 min after washing with 1X TBST. Finally, the immunoreactive bands were visualized by chemiluminescence detection.
3
Cytosolic and Nuclear Protein Extraction for Western Blot Analysis
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A RIPA buffering system was used to acquire the cell lysate neurons. Cytosol protein and nuclear protein were extracted using the Cytosol Protein Extraction kit (Beyotime) and Nuclear Protein Extraction kit (Invitrogen), respectively. Bicinchoninic acid (BCA) method was carried out to determine the concentrations of protein samples. SDS-PAGE was employed to separate the proteins, which were then electronically transferred to polyvinylidene fluoride (PVDF) membranes. After blocking, primary antibodies against Wnt5a, phosphorylated JNK1 (p-JNK1), JNK1, cleaved caspase3 (c-caspase3), IL6, TNFα, and GAPDH were used to incubated the membranes loaded with cytosol protein samples at 4°C for 10 h. Specific antibodies against p65 and histone H3 were used to incubate the membranes loaded with nuclear proteins. Membranes were washed by TBST (0.02%) and then incubated with corresponding secondary antibodies at room temperature for 20 min. The immunoblots were visualized by ImageQuant LAS 4000 after being developed by ECL reagents.
4
Protein Extraction and Analysis Protocol
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Tissues or cells were lysed in RIPA lysis buffer (Beyotime, China). For membrane protein isolation, cells were lysed by membrane and Cytosol Protein Extraction Kit, according to the manufacture’s instruction (Beyotime). Proteins extracted from tissues or cells were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA), and then incubated with the primary antibody followed by anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase (Abcam, Cambridge, MA, USA). The primary antibodies used were as follows: anti-TrkB (ab18987), anti-Akt (ab32505), anti-p-Akt (ab81283), anti-Bax, anti-Bcl-2anti-ADRB2 (ab182136), anti-PKA (ADI-KAS-PK017-F; Enzo, Hong Kong, China), anti-p-PKA (ab75991), and anti-Caveolin-3 (ab2912). β-actin (6008-1-Ig, ProteinTech, Rosemont, IL, USA) was used as an endogenous control for total protein. Na/K ATPase (sc-514614, Santa Cruz, USA) was used as an endogenous control for membrane protein. All antibodies were obtained from Abcam unless otherwise stated.
5
NEFA-Induced Cytosolic Protein Redistribution
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The hepatocytes were treated with various NEFA concentrations for 4 h following pretreatment with the antioxidant NAC or with an inhibitor of JNK (SP600125) or ERK (PD98059). To detect the redistribution of cyt c and AIF, the proteins in the cytosolic fraction were isolated using a cytosol protein extraction kit (Beyotime Institute of Biotechnology, Nanjing, China) according to the multiple centrifugation method. The protein concentrations were measured using the BCA method (Applygen, Beijing, China).
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