Briefly, total protein extracts were prepared from homogenized tissues or cultured cells with RIPA buffer containing protease inhibitors and a phosphatase inhibitor (cOmplete & PhosSTOP, Roche, Basel, Switzerland). Protein was quantified with an Enhanced BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were separated by SDS-PAGE before wet transfer onto nitrocellulose membranes (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The membranes were blocked with 5% nonfat milk for 2 h at room temperature. After blocking, the membranes were incubated at 4 °C overnight with primary antibodies against SKP2 (1:1000; ab183039, Abcam, Cambridge, UK), GLUT1 (1:1000; 66,290–1, Proteintech, Rosemont, IL, USA), or β-actin (1:5000; sc47778, Santa Cruz, AR, USA), the latter of which was used as an internal control. Protein complexes were developed and observed with a chemiluminescent detection system (Millipore, Waltham, MA, USA) and a G-Box Chemiluminescence image capture system (Syngene, Cambridge, UK). The resulting bands were analyzed using Gel-Pro Analyzer software (Media Cybernetics).
Gel pro analyzer software
Manufactured by Media Cybernetics
Sourced in United States, Germany, China
The Gel-Pro Analyzer software is a laboratory analysis tool designed for the visualization and quantification of gel electrophoresis data. It provides users with the ability to analyze and interpret gel images, including features such as band detection, molecular weight calculation, and densitometric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (20 mentions)
Bca protein assay kit, by Beyotime (9 mentions)
Ripa buffer, by Beyotime (5 mentions)
Chemilux imager csx 1400m, by Intas (5 mentions)
Gapdh, by Abcam (4 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Immobilon p, by Merck Group (4 mentions)
Nitrocellulose membrane, by GE Healthcare (4 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (4 mentions)
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
Sc 47778, by Santa Cruz Biotechnology (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Bio dot apparatus, by Bio-Rad (3 mentions)
Ipvh00010, by Merck Group (3 mentions)
β actin, by Merck Group (3 mentions)
Ripa buffer, by Merck Group (3 mentions)
Ab183039, by Abcam (2 mentions)
Chemiluminescent detection system, by Merck Group (2 mentions)
Complete phosstop, by Roche (2 mentions)
154 protocols using gel pro analyzer software
1
Western Blot Analysis of Protein Expression
2
RhoA Activation Assay Protocol
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RhoA activation assay reagents were purchased from Cytoskeleton and used as per the manufacturer’s instructions. Briefly, GTP-bound active RhoA was pulled down with Rhotekin-RBD beads for 1 h at 4 °C, and then subjected to immunoblot analysis. Immunoblots of whole-cell lysates were used to assess for total RhoA. The IOD of bands was evaluated by densitometry and analyzed by using Gel-Pro Analyzer software (Media Cybernetics).
3
Protein Expression Analysis in LX-2 Cells
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Total protein was isolated from LX‐2 cells using RIPA (Solarbio) and quantified using a BCA Protein Assay Kit (Solarbio). Then equal amounts of protein samples were separated on SDS‐PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk for 60 minutes, and incubated with primary antibodies against CAPRIN1 (1:500; Proteintech), Cyclin D1 (1:1000; Bioss, Beijing, China), Cyclin E1 (1:1000; Proteintech), p‐RbS807 (1:1000; Bioss), TGF‐β2 (1:1000; Bioss), COL1A1 (1:1000; Bioss), α‐SMA (1:1000; Bioss), p‐Smad2ser467 (1:1000; Cell Signaling Technology, Trask Lane Danvers, MA), Smad2 (1:1000; Cell Signaling Technology), p‐Smad3S423/S425 (1:1000; Cell Signaling Technology), Smad3(1:1000; Cell Signaling Technology) and β‐actin (1:1000; Santacruz Biotechnology) at 4°C overnight. Goat anti‐rabbit IgG or goat anti‐mouse secondary antibodies (1:3000; Solarbio) were employed at 37°C for 60 minutes. The bands were visualized using electro‐chemi‐luminescence (ECL) Western Blotting Substrate (Solarbio). The protein expression was standardized to β‐actin and quantified using Gel‐Pro‐Analyzer software (Media Cybernetics, Rockville, MD).
4
Western Blot Analysis of PIAS3 and STAT3
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After transfection for 24 hours, cells were lyzed in lysis buffer (Beyotime, Shanghai, China) with the protease inhibitor (Roche, Mannheim, Germany). The protein concentration was examined with a BCA Protein Assay kit (Pierce, Rockford, IL). For sodium dodecyl sulphate polyacrylamide (SDS) gel electrophoresis, equal quantities of total protein (30 μg) were separated on 10% SDS‐PAGE and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). After blocking in nonfat milk at room temperature for 1 hour, the membranes were incubated with the specific antibodies against PIAS3 (Abcam, Cambridge, MA), STAT3 (Cell Signalling Technology, Beverly, MA), p‐STAT3 (Tyr705) (Cell Signalling Technology) and β‐actin (Cell Signalling Technology) at 4°C overnight. After washing with Tris‐buffered saline‐tween (TBS‐T), the membranes were then incubated with horseradish peroxidase (HRP)‐conjugated secondary antibody at room temperature for 1‐2 hours. Protein bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Pierce). The Western blots were quantified using Gel‐Pro analyzer software (v4.5, Media Cybernetics, Rockville, Maryland, USA).
5
Protein Expression Analysis by Western Blot
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Biopsies were kept stored at − 80 °C and prepared for analysis on the same day. To measure protein expression, total protein concentration was measured by the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). 40 μg protein samples were loaded and separated on a 5–10% SDS-PAGE gradient gel, and electrically transferred onto a PVDF membrane (Millipore, Billerica, USA). PVDF membranes were blocked with 5% nonfat milk in Tris buffer solution containing 0.1% Tween-20 (TBST) for 1 h at room temperature, followed by incubation with primary antibody. After washing four times with TBST (5 min each, room temperature), the membranes were incubated for 1 h with fluorescent-labeled secondary antibodies (anti-Rabbit IgG at 1:5000). Then, the membranes were washed and proteins were visualized by using an enhanced peroxidase/luminal chemiluminescence reaction (ECL Western blotting detection reagent) (Wanleibio, China), and the final images were analyzed by densitometry using Gel-Pro-Analyzer software (Media Cybernetics, USA). All primary and secondary antibodies were purchased from Wanleibio Biology Ltd., Shenyang, China.
6
Endo III Product Analysis by PAGE
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To analyze the products formed by Endo III, the substrates were 5′-32P-labeled with phage T4 polynucleotide kinase and γ-32P-ATP; the reaction was carried out under the conditions described above. The products were precipitated by adding 10 volumes of 2% LiClO4 in acetone. The precipitates were washed three times with 100 μL of acetone, dried, dissolved in 4 μL of water and 3 μL of loading buffer (7 M urea, 0.1% bromophenol blue, and 0.1% xylene cyanol), and analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) in a 20% gel. The gels were visualized using Agfa CP-BU X-ray film (Agfa-Geavert, Mortsel, Belgium), and the autoradiograms were scanned and quantified in the Gel-Pro Analyzer software (Media Cybernetics, Rockville, MD, USA).
7
Western Blot Quantification Protocol
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Protein concentrations were measured using the Bio-Rad Bradford protein assay. The proteins were separated by 12% SDS-polyacrylamide gel electrophoresis, transferred to IMMOBILON PVDF membranes (Millipore) and incubated overnight at 4°C in blocking buffer containing 2% ECL blocking reagent (GE Healthcare) and 0.1% Tween 20 in PBS. The membranes were probed with either anti TFPI-2 (1:500, SantaCruzBiotechnology) or anti α-tubulin (1:1000, Sigma-Aldrich) antibody for 1h at room temperature. Antibody binding was detected with anti-mouse IgG-HRP (1:10,000, Sigma Aldrich) for 45min at room temperature. The signals were detected with ECL Prime (GE-Healthcare). Densitometry of protein bands was analysed with GelPro Analyzer Software (Media Cybernetics).
8
Fatty acid metabolism in rodents
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Reagents and kits were purchased from Sigma, except for the following: [1‐14C]‐palmitic acid (PerkinElmer Life Sciences, Santa Clara, CA, USA), liquid scintillation cocktail (LSC, PerkinElmer Life Sciences), [carboxyl‐14C]‐triolein (PerkinElmer Life Sciences), Man–Rogosa–Sharpe (MRS, Becton Dickinson, NJ, USA), orlistat (Xenical, Roche, Basel, Switzerland), EnzyChrom™ Free Fatty Acid Assay Kit (BioAssay Systems, Hayward, CA, USA). The sterilizable 384‐well plate and 384‐pin replicator were from Nunc. The membrane semidry system was from Bio‐Rad, and the X‐ray film was from Kodak. The gel‐pro analyzer software was from Media cybernetics. The magnetic resonance imaging (MRI) images were obtained with a Bruker Biospec 47/40 4.7‐Tesla instrument (Bruker, Billerica, MA, USA) and analyzed with image j software (NIH, Bethesda, MD, USA). The serum was analyzed with a Rat/Mouse ELISA kit (LINCO research, St. Charles, MO, USA), a Leptin ELISA kit (R&D System, Minneapolis, MN, USA), a blood glucose meter (Accu‐Chek, Roche) and Cholesterol ELISA kits (Asan Pharmaceutical, Seoul, Korea).
9
Western Blot Analysis of VEGF Signaling
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Cells in each group were lysed with NP-40 lysate (Beyotime Institute of Biotechnology) including 1% phenylmethanesulfonyl fluoride (PMSF). The concentration of total proteins was quantitated by a bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology). Then equal amounts (20 µg) of different proteins were loaded and separated using SDS-polyacrylamide gel electrophoresis (PAGE) followed by electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After being blocked with 5% non-fat milk at 4°C overnight, the membranes were probed with specific primary antibodies against VEGFR-2, p-VEGFR-2 (Tyr1175), ERK1/2, p-ERK1 (pT202/pY204) + p-ERK2 (pT185/pY187) (all 1:1,500 diluted); and VEGF-A, MEK1, p-MEK1 (pS298) (all 1:1,000 diluted; Abcam, Cambridge, MA, USA Abcam) at 4°C overnight. The membranes were then incubated with the secondary goat anti-rabbit IgG-HRP antibody (1:20,000 diluted; Wuhan Boster Biological Technology, Ltd., Wuhan, China) at 37°C for 40 min. The unbound antibodies in each step were washed with TBST for three times. The target bands were visualized by an enhanced chemiluminescence (ECL; Millipore), and the protein intensities were detected by Gel-Pro Analyzer software (Media Cybernetics, Inc., Bethesda, MD, USA). GAPDH was used as an internal control.
10
Protein Expression Analysis by Western Blot
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Cell pellet was lysed in RIPA buffer [50 mm Tris-HCl (pH 8.0), 0.15 m NaCl, 1% Triton X-100, 0.5% NaDoc, 0.1% SDS, 1 mm EDTA, 1 mm EGTA, 1 mm PMSF (Amersco) and 1 μg/ml Pepstatin A (Sigma) protease inhibitors]. The cells were then sonicated using BioruptorTMUCD-200 (Diagenode) for 15 min at a low speed. Cell lysates were centrifuged for 20 min at 15 000g at 4 °C, and the protein supernatant was collected. Protein samples were loaded on a 10% (w/v) Tris-HCl SDS-PAGE gels for electrophoresis, transferred to PVDF membrane (Millipore), blotted, and then probed with anti-AID (CST, #4959), anti-BCL6 (Abcam, #ab33901), anti-DNMT1 (Abcam, #ab13537), anti-DNMT3A (Abcam, #ab2850), anti-DNMT3B (Abcam, #b2850), anti-Caspase 3 (Abcam, #ab13586) antibodies. Anti-GAPDH (Abcam, #ab9485) was used as a loading control. The signal was further detected using the secondary antibody of goat anti-Rabbit, goat anti-Rat, goat anti-Mouse IgG conjugated with horseradish peroxidase (Thermo Fisher). Band signal was visualized by Immobilon™ Western Chemiluminescent HRP substrate (Millipore). Integrating optical density was analyzed using gel-pro analyzer software (Media Cybernetics) and ratios of bands were all referenced to the loading control.
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