Tissues were homogenized using lysis buffer (keyGEN Bio TECH, China), containing protease and phosphatase inhibitors and centrifuged at 12,000 × g, 4°C for 5 min. The supernatant was collected where protein concentration was measured using the BCA Protein Assay kit (keyGEN Bio TECH, China), according to the manufacturer's instructions. The SDS-PAGE protein loading buffer (5×) (Beyotime, China) was added to the sample and heated at 100°C for 5 min to fully denature the protein. Proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane and then blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween 20 and incubated overnight at 4°C with primary antibodies targeting phosphorylated hormone sensitive lipase (p-HSL) (ser563) (1 : 1000, #4139, Cell Signaling Technology, USA), hormone sensitive lipase (HSL) (1 : 1000, ab45422, abcam, USA), adenosine 5'- monophosphate- (AMP-) activated protein kinase (AMPK) (1 : 1000, ab3760, abcam, USA), and phosphorylated AMPK (p-AMPK) (1 : 1000, ab133448, abcam, USA). Then, the blots were incubated with HRP-conjugated secondary antibodies and detected by Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA) using Molecular Imager ChemiDOC™ XBS imaging systems (Bio-Rad Laboratories).
Sds page protein loading buffer 5
Manufactured by Beyotime
Sourced in China
SDS-PAGE protein loading buffer (5×) is a concentrated solution used to prepare protein samples for separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The buffer contains SDS, a detergent that denatures proteins and gives them a uniform negative charge, as well as other components that facilitate sample preparation and loading onto the gel.
Automatically generated - may contain errors
Lab products found in correlation
Ab45422, by Abcam (1 mentions)
Ab133448, by Abcam (1 mentions)
Ab3760, by Abcam (1 mentions)
Molecular imager chemidoc xbs imaging systems, by Bio-Rad (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Lysis buffer, by Keygen Biotech (1 mentions)
Bca protein assay kit, by Keygen Biotech (1 mentions)
Acacetin, by MedChemExpress (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Matrigel basement membrane matrix, by Corning (1 mentions)
Yeast extract, by Thermo Fisher Scientific (1 mentions)
Sds page gel preparation kit, by Beyotime (1 mentions)
Bca protein concentration determination kit, by Beyotime (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Gapdh, by Signalway Antibody (1 mentions)
Cleaved caspase 3, by Signalway Antibody (1 mentions)
4 protocols using sds page protein loading buffer 5
1
Protein Analysis of Lipid Metabolism Regulators
2
Acacetin-Induced Antiproliferative Effects
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RPMI-1640 was purchased from Gibco, 2457402; Fetal bovine serum was purchased from Viva cell, 2209058; 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazole bromide (MTT) was purchased from Suolarbio, M8180; Dimethyl sulfoxide (DMSO) was purchased from Mreda, M1538; Propidium iodide of cell cycle was purchased from Invitrogen, 2238888; RNase was purchased from ATRANS, GE101-01; The cell apoptosis kit was purchased from Suolarbio, CA1020; The matrigel basement membrane matrix was purchased from Corning, 356234; SDS-PAGE protein loading buffer (5×) was purchased from Beyotime, P0015L; Acacetin was purchased from MedChemExpress (MCE), HY-N0451.
3
Berberine Identification and Characterization
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Canagliflozin, manufactured by Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China) Berberine, which is extracted from coptis root and identified through nuclear magnetic resonance (NMR) analysis, has been confirmed as berberine. This experiment employed protein peptone (Oxoid, Hampshire, UK), yeast extract (Oxoid, Hampshire, UK), crystal violet (Beyotime, Shanghai, China), the SDS-PAGE gel preparation kit (Beyotime, Shanghai, China), SDS-PAGE protein loading buffer (5×) (Beyotime, Shanghai, China), Coomassie brilliant blue ultra-fast staining solution (Beyotime, Shanghai, China), pre-stained protein marker VII (8–195 kDa) (Savier, Wuhan, China), and the BCA protein concentration determination kit (Beyotime, Shanghai, China). Unless otherwise specified, all other reagents used in the experiment were commercially purchased.
4
Western Blotting of Cell Signaling Proteins
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Western blotting was performed as described in a previous study with some modifications [20 (link)]. Protein extracts from cells or hippocampi were prepared in a modified RIPA buffer supplemented with protease inhibitors (200612, Signalway Antibody, Greenbelt, MD, USA). The BCA method was used to determine the concentration of protein. The protein extracts were boiled after being diluted in SDS-PAGE protein loading buffer (5×) (Beyotime, Shanghai, China) at a ratio of 4:1. Following separation, the proteins were transferred to polyvinylidene difluoride membranes and blocked in TBST buffer (20 mM Tris–HCl, pH 7.4, 137 mM NaCl, and 0.1% Tween-20) with 5% non-fat milk at 37 °C for 1 h, and incubated at 4 °C with primary rabbit polyclonal antibodies (cleaved caspase-3, 49500, 1:500, Signalway Antibody; BCL-W, 40641, 1:1000, Signalway Antibody; SAPK/JNK (pThr183), 11249, 1:500, Signalway Antibody; SMAC/DIABLO, 39330, 1:500, Signalway Antibody; GAPDH, 21612, 1:3000, Signalway Antibody) overnight. After extensive rinsing, the membranes were incubated with the appropriate HRP-conjugated secondary antibody, then visualized using Super ECL Plus reagents. The gray values of the protein bands were quantified by the optical density using ImageJ software (1.41v, US National Institutes of Health, Bethesda, MD, USA).
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