Liver tissue collected at the end of the hyperinsulinemic-euglycemic clamp was snap frozen in liquid nitrogen and stored at −80°C. Liver tissue was cut on dry ice and homogenized in a RIPA buffer. Samples were run on SDS-PAGE gel and transferred overnight onto nitrocellulose membranes. Total protein was assessed via REVERT stain (Li-COR Biosciences, cat # 926-11011) and was used to normalize target signals [AKT and phosphor-AKT(ser473)]. Nitrocellulose membranes were then blocked in 2% Bovine Serum Albumin for 1 h before being incubated with a primary antibody. Primary antibodies used were P-AKT: Cell Signaling Technology, Catalog # 4060S (Danvers, MA, USA) (rabbit) and AKT (pan): Cell Signaling Technology, Catalog # 2920S (mouse) (Danvers, MA, USA). Blot was then washed in TBST before incubation with a secondary antibody. Blots were imaged using Li-COR scanner and quantified using Image Studio. All protein targets were normalized to total protein.
Revert stain
Manufactured by LI COR
Sourced in Germany
REVERT stain is a reversible protein stain designed for visualization of proteins in polyacrylamide gels. It can be easily removed from the gel, allowing for subsequent analysis or further processing of the proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bradford assay, by Thermo Fisher Scientific (2 mentions)
Wet transfer method, by Bio-Rad (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Odyssey clx imaging system, by LI COR (2 mentions)
Revert wash buffer, by LI COR (2 mentions)
Pan akt, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by LI COR (1 mentions)
Fluorescently conjugated secondary antibodies, by LI COR (1 mentions)
Phosphorylated akt s473, by Cell Signaling Technology (1 mentions)
Blocking buffer, by LI COR (1 mentions)
Phosphorylated gsk 3β s9, by Cell Signaling Technology (1 mentions)
Image studio, by LI COR (1 mentions)
Mouse anti hur, by Santa Cruz Biotechnology (1 mentions)
Mouse anti stat3, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Amersham hybond, by Cytiva (1 mentions)
Odyssey fc scanner, by LI COR (1 mentions)
Rabbit anti p53, by Cell Signaling Technology (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
9 protocols using revert stain
1
Liver Insulin Signaling Pathway Analysis
2
Quantitative Western Blot Analysis in Metabolic Tissues
3
Quantitative Western Blotting Analysis
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Western blotting was performed by standard procedures using Novex Tris-Glycine buffered gradient WedgeWell gels (8–16%) (Invitrogen – Thermo Fisher Scientific) in a Mini Cell apparatus. All protein transfers were performed overnight onto Amersham™ Hybond™ (Cytiva) PVDF membranes 0.45 µm pore size. Chemiluminescence from HRP-conjugated secondary antibodies (1:20.000 dilution) (Thermo Fisher Scientific) was developed using SuperSignal™, West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Antibodies used: Mouse anti-Streptag II antibody (Antibodies Online) ABIN7250959, (1:1.000) Mouse anti-STAT3 (Cell signaling technologies, CST-9139) (1:1000), Mouse anti-HuR (Santa Cruz Biotechnology) (SC-365338) (1:5000), Rabbit anti-p53 (Cell signaling technologies, CST-2527) (1:500), and Rabbit anti-HIPK3 (Abcam Ab72538) (1:500). Acquisition and quantifications of band intensities were performed using a Licor™ OdysseyFc™ scanner and Image Studio version 5.2 software. Band intensities were normalized to abundant house-keeping protein HuR or in some cases total protein assessed by Revert Stain (Licor™). The normalized band intensities are based on 3 biological replicates (quantified western blots - plotted data). Error bars represents standard deviations from means (n=3).
4
Western Blot Protein Analysis
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Cells were lysed using RIPA buffer (Thermo Fisher) complemented with protease and phosphatase inhibitors (Thermo Fisher). Lysates were sonicated in short bursts and centrifuged at 15,000 rpm for 15 min. Protein concentration was measured using Bradford assay (Thermo Fisher). 20–50 ug of total protein was loaded per sample and separated on a 4–15% SurePAGE gradient gel (GenScript). The proteins were transferred onto nitrocellulose membrane using Trans-Blot Turbo Transfer System (Bio-Rad). REVERT stain (Li-COR) was used to normalize for total protein loaded. Membranes were blocked with PBS Odyssey Blocking Buffer (Li-COR) for 1 h at room temperature and incubated overnight at 4°C with primary antibodies, followed by secondary antibodies for 1 h at room temperature. Both primary and secondary antibodies were listed in Supplementary Table S1 . Images were obtained using an Odyssey FC or CLX imaging system.
5
Quantitative Western Blotting Analysis
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Lysates were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Total protein was quantified using REVERT stain (Li-Cor Biosciences). Primary antibodies were used according to manufacturer recommendations: anti-MUC16 [X75] (Novus Biologicals, NB6001468), anti-human cathepsin D (Cell Signaling, #2284), anti-mouse cathepsin D (Novus Biologicals, AF1029), anti-cathepsin B [D1C7Y] (Cell Signaling, #31718), anti-cathepsin L [33/2] (Santa Cruz, sc-32320), anti-LAMP1 [1D4B] (Developmental Studies Hybridoma Bank), anti-VDAC1 (Cell Signaling, #4661), anti-GOLGIN-97 (Cell Signaling, #13192), anti-CALR (Cell Signaling #12238), and anti-CATLASE (Cell Signaling, #14097). Secondary antibodies were used according to manufacturer recommendations.
6
Western Blot Analysis of Hippocampal Proteins
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Hippocampal lysates were denatured at 95°C using the 2-Mercaptoethanol based loading buffer. Proteins were separated using the SDS-polyacrylamide gel electrophoresis and western blot procedure. Custom made 8-10% gels were used to run the samples for 1.5 hours at 110 voltage. The proteins were then transferred to a precut 0.2 µm nitrocellulose membrane (Cat: 1620146; Biorad, USA) using a wet transfer method (Biorad). Later the membranes were incubated in a blocking solution containing 0.5% bovine serum albumin (Art. No. 8076.4, Roth, Germany), 1xTBS, and 0.1%Tween-20 for 30 minutes at room temperature on a rotating shaker. The membranes were then incubated with primary antibodies overnight at 4°C with gentle agitation. On the second day, the membranes were rinsed with TBS-Tween solution 3 x 5 minutes, followed by a 1-hour incubation with the uorescently conjugated secondary antibodies, rinsed again and air-dried covered by aluminum foil. The proteins were detected using the Omega Lum (Labgene, CH). The optical density of the protein bands was determined using Image J software and normalized to the Beta-actin control. For an accurate total protein quanti cation REVERT stain (LI-COR Biosciences-GmbH, Germany) was also employed.
7
Western Blot Analysis of Hippocampal Proteins
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Hippocampal lysates supplemented were denatured at 95°C using the 2-Mercaptoethanol based loading buffer. Proteins were separated using the SDS-polyacrylamide gel electrophoresis and western blot procedure. Custom made 8-10% gels were used to run the samples for 1.5 hours at 110 voltage. The proteins were then transferred to a precut 0.2 µm nitrocellulose membrane (Cat: 1620146; Biorad, USA) using a wet transfer method (Biorad). Later the membranes were incubated in a blocking solution containing 0.5% bovine serum albumin (Art. No. 8076.4, Roth, Germany) , 1xTBS, and 0.1%Tween-20 for 30 minutes at room temperature on a rotating shaker. The membranes were then incubated with primary antibodies overnight at 4°C with gentle agitation. On the second day, the membranes were rinsed with TBS-Tween solution 3 x 5 minutes, followed by a 1-hour incubation with the fluorescently conjugated secondary antibodies, rinsed again and air-dried covered by aluminum foil. The proteins were detected using the Omega Lum (Labgene, CH). The optical density of the protein bands was determined using Image J software and normalized to the Beta-actin control. For an accurate total protein quantification REVERT stain (LI-COR Biosciences-GmbH, Germany) was also employed.
8
Quantitative Total Protein Estimation
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Total protein standards were prepared by diluting 5 mg/mL bovine serum albumin (BSA) in DMD lysis buffer, and then diluting serially 1:2 six times (5–0.078 mg/mL). Test samples were thawed on ice. Aliquots were removed for total protein measurement. A total of 1 μL of each test sample or BSA standard was pipetted onto a nitrocellulose membrane, at least in duplicate measurement. After air drying, the membrane was incubated with 1X LI-COR REVERT™ stain (LI-COR, Lincoln, NE) as per the vendor protocol. The membrane was incubated in the stain for 5 min, and then washed twice in equal volumes of 1X LI-COR REVERT™ Wash Buffer. The dots on the membrane were visualized and quantitated on a LI-COR Odyssey CLX™ Imaging System (LI-COR, Lincoln, NE). Total protein on the samples was determined by interpolation of dot-blot fluorescence intensities into the BSA standard curve as described previously (36 (link), 37 (link)).
9
Quantitative Total Protein Estimation
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