Total proteins from cells or myocardial tissue were extracted and quantified. Then, 30–50 μg of each sample was separated using 10% SDS-PAGE gel electrophoresis (stacking gel 50 V, separating gel 100 V), and transferred to a nitrocellulose membrane (100 V, 75 min). Membranes were then blocked and incubated with primary antibodies followed by secondary antibodies. HRP-labeled secondary antibodies were detected using chemiluminescence, and the grayscale of the protein bands was analyzed using Gel-pro Image Analysis Software (Media cybernetics, Rockville, MD, USA).
Gel pro image analysis software
Manufactured by Media Cybernetics
Sourced in United States
Gel-pro Image Analysis Software is a computer program designed for the analysis of gel electrophoresis images. It provides tools for visualizing, measuring, and quantifying bands or spots within the gel image.
Automatically generated - may contain errors
Lab products found in correlation
P irs, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
β actin, by Media Cybernetics (1 mentions)
P ampkα thr172, by Cell Signaling Technology (1 mentions)
Supersignal west dura extended duration substrate kit, by Thermo Fisher Scientific (1 mentions)
Ampkα, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
β actin, by ZSGB-BIO (1 mentions)
Super signal detection kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using gel pro image analysis software
1
Quantitative Protein Analysis of Cells
2
Western Blotting of MAPK Signaling
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Western blotting was performed as described previously (Zhang et al., 2015 (link)). Briefly, total protein in CMECs was extracted and quantified. Next, equal amounts of denatured protein samples (20–50 μg) were subjected to 8% SDS-PAGE and probed with corresponding primary antibodies for Egr-1 (1:1500), p-JNK (1:1000), total-JNK (1:2000), p-ERK1/2 (1:2000), total-ERK1/2 (1:2000), p-p38 (1:2000), total-p38 (1:2000), and β-actin (1:4000) at 4°C overnight, followed by secondary antibodies [HRP-labeled rabbit anti-mouse IgG (1:50,000) or HRP-labeled goat anti-rabbit IgG (1:80,000)]. HRP-labeled secondary antibodies were detected by chemiluminescence, and the protein bands were analyzed using Gel-pro Image Analysis Software (Media Cybernetics, USA). The ratio of p-JNK/JNK, p-ERK1/2/ERK1/2, and p-p38/p38 reflected the activation of JNK, ERK1/2, and p38, respectively, and the ratio of Egr-1/β-actin represented the expression of Egr-1.
3
Western Blot Analysis of Inflammasome Proteins
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The cells were lysed with RIPA solution, and the total cell proteins in each group were extracted. The BCA protein detection kit was used to measure the protein concentration of each sample to ensure that the loading total protein in each group was the same. After SDS-PAGE separation, the proteins were transferred onto the nitrocellulose (NC) filter membrane. After the electric transfer, the membrane was sealed with 5% skim milk for 1 h. After sealing, membrane was treated with P2X7 (1:300), NLRP3 (1:300), ASC (1:100), Caspase-1 (1:100), and GSDMD1 (1:3,000) antibodies and incubated overnight at 4°C. Then the membranes were washed with TBST for 3 times, 10 min each time. The goat anti-rabbit-IgG-HRP antibody (1:20,000) and goat anti-murine-IgG-HRP antibody (1:20,000) diluted in 5% skim milk were added, and incubated at room temperature for 1 h. The membranes were washed again with TBST for 3 times, 10 min each time. Finally, chemiluminescent solution was used for exposure in a dark room. Gel-Pro image analysis software (Media Cybernetics, United States) was used to analyze the optical density of the protein bands.
4
Western Blot Analysis of Protein Levels
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Proteins (50 μg) from lysed cells were separated by 10% SDS‐PAGE and transferred to nitrocellulose membranes. After blocking for 2 hours, the membranes were incubated overnight with primary antibodies followed by horseradish peroxidase (HRP)‐conjugated secondary antibodies. The protein bands were visualized using enhanced chemiluminescence. Densitometry analysis of protein levels was performed with Gel‐pro Image Analysis Software (Media Cybernetics, Rockville, MD, USA).
5
Western Blot Analysis of Protein Signaling
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Total proteins were extracted and quantified from the cell or from the retroperitoneal and epididymal adipose tissue. Then, 30–50 μg protein was isolated from each sample using 10% SDS-PAGE (stacking gel, 50 V; separating gel, 100 V), and transferred into the nitrocellulose membrane (100 V, 75 min). Membranes were blocked and incubated with primary antibodies followed by secondary antibodies. Horseradish peroxidase-labeled secondary antibodies were detected by chemiluminescence and the grayscale of the protein bands was analyzed with Gel-pro Image Analysis Software (Media Cybernetics, Rockville, MD, USA). The primary antibodies against p-IRS (Cell Signaling Technology, Danvers, MA, USA), p-Akt (Cell Signaling Technology, Danvers, MA, USA), and beta-Actin (Cell Signaling Technology, Danvers, MA, USA) were used in this study.
6
Western Blot Analysis of Protein Markers
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H9c2 cells were washed with cold PBS three times, and scraped into mixed lysate buffer containing RIPA buffer and phenylmethanesulfonyl fluoride (PMSF; 1,000:1). Lysates were centrifuged for 15 min at 12,000 × g at 4°C. Protein was quantified using a BCA protein assay kit (Pierce, Rockford, IL, United States), and 25 μg of each sample was resolved on 10% SDS-PAGE (stacking gel 50 V, separating gel 100 V), and transferred to nitrocellulose membranes (100 V, 75 min). Then, membranes were blocked with 5% fat-free milk, and then incubated overnight at 4°C with primary antibody (Egr-1—1: 1,000, SIRT1—1: 1,000, Ac-FOXO1—1:200, FOXO1—1: 1,000 and β-actin—1: 3,000), followed by incubation with HRP-conjugated goat anti-rabbit IgG (1: 20,000) or HRP-labeled goat anti-mouse IgG (1: 30,000) for 1 h at room temperature. Protein bands were analyzed with Gel-pro Image Analysis Software (Media cybernetics, United States).
7
Apoptosis Regulation via AMPK/Sirt1 Pathway
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The expression of the apoptosis-related proteins (Bcl-2 and Bax) and AMPK/Sirt1 were determined using Western blotting. Briefly, equivalent protein samples from each group were separated using 10% or 12% SDS-PAGE and then transferred onto nitrocellulose membranes. The membranes were blocked using 5% skim milk at RT for 1 h and incubated with primary antibodies against p-AMPKα (Thr172) (no. 2535, 1:1000, Cell Signaling Technologies, MA, USA), Bcl-2 (no. 26539-1-AP, 1:2000, Proteintech, Wuhan, Hubei, China), AMPKα (no. 2603, 1:1000, Cell Signaling Technologies, MA, USA), Bax (no. 2772, 1:1000, Cell Signaling Technologies, MA, USA), Sirt1 (no. 9475, 1:1000, Cell Signaling Technologies, MA, USA), and β-actin (no. TA09, 1:2000, ZSGB-BIO, Beijing, China) at 4 °C overnight. The membranes were rinsed thrice with Tris-buffer saline with 0.25% Tween-20 (v/v) and incubated with a horse radish peroxidase (HRP)-conjugated secondary antibody (no. BA1054, BA1050, 1:30000, Boster Biological Technology, Wuhan, China). The membranes were visualized using a SuperSignal™ West Dura Extended Duration Substrate kit (Thermo Fisher Scientific, IL, USA). After exposing the X-ray film, the density of the bands was standardized over β-actin and calculated using a Gel-Pro Image Analysis software (Media Cybernetics, Rockville, MD, USA).
8
Immunoblotting Analysis of Mitochondrial Proteins
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The protocol of extracting cells is as previously described [5 (link), 20 (link)]. The equal denatured protein underwent 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then was transferred to a nitrocellulose membrane by electrophoresis. Membranes were blocked with 5% fat-free milk and immunoblotted with diluted primary antibody: anti-phospho-JNK (1 : 400), anti-Sab (1 : 250), anti-p-Src (1 : 250), anti-c-Src (1 : 1000), anti-COX-IV (1 : 400), and anti-β-actin (1 : 5000) and then incubated at 4°C overnight, which was followed by exposure to horseradish peroxidase-conjugated secondary antibodies (typically 1 : 20,000 dilution) for 1 hour at room temperature. Transferred proteins were visualized by use of a SuperSignal detection kit (Thermo-Fisher Scientific, Waltham, MA, USA). The bands were analyzed with the Gel-Pro Image Analysis Software (Media Cybernetics, USA). The level of each mitochondrial protein was expressed as its density to the density of mitochondrial loading control COX-IV. To avoid the error in defferent batches, the desity ratio between interest protein and COX-IV in the H/R group was set as 1.
9
Western Blotting Quantification of Signaling Proteins
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Western blotting was performed as described previously10 . Briefly, total proteins were extracted and quantified. Next, 30–50 μg of each sample was separated using 10% SDS-PAGE gel electrophoresis (stacking gel 50 V, separating gel 100 V), and transferred to nitrocellulose membrane (100 V, 75 min). Membranes were then blocked and incubated with primary antibodies (Egr-1, p-JNK, JNK, and β-actin at 1:1,000, 1:200, 1:2,000, and 1:3,000 dilutions, respectively) followed by secondary antibodies (HRP-labeled rabbit anti-goat IgG, HRP-labeled goat anti-rabbit IgG, IRDyeTM 800CW goat anti-rabbit IgG, or IRDyeTM 800CW goat anti-mouse IgG at dilutions of 1:20,000, 1:30,000, 1:10,000, or 1:10,000, respectively); IRDye incubations were performed in the dark. HRP-labeled secondary antibodies were detected using chemiluminescence, and the grayscale of the protein bands was analyzed using Gel-pro Image Analysis Software (Media cybernetics, USA). IRDye-labeled secondary antibodies were analyzed using an Odyssey infrared imaging system (Odyssey LI-COR, USA). The ratio of p-JNK/JNK represented the activation of JNK, JNK/β-actin indicated the overall expression of JNK, and the ratio of Egr-1/β-actin reflected the expression of Egr-1.
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