Isolated rat colonocytes were lysed in RIPA buffer containing a protease inhibitors cocktail (Roche). Total protein extracts (30 μg) were loaded into 4–12% Criterion XT gel (Bio-Rad) before electrophoresis in MOPS buffer (Bio-Rad). After transfer on nitrocellulose membrane and incubation in blocking solution (TBS pH 7.5, 0.05% Tween 20 and 5% (weight:volume) BSA, membranes were incubated overnight (4 °C) with a primary antibody directed against activated-caspase 3 (Abcam 2303, rabbit, 1/1000) or proliferating cell nuclear antigen (PCNA, Abcam 29, mouse, 1/1000) or claudin-1 (Invitrogen, 717800, rabbit, 1/250) diluted in the blocking solution. After washes, blots were incubated for 2 h at room temperature with an anti-rabbit or anti-mouse HRP-linked secondary antibody (Jackson Immuno Research Laboratories, 1/5000) or a goat anti-actin-HRP (Santacruz Biotechnologies C-11, 1/1000) diluted in the blocking solution. After 3 washes, detection was performed by chemiluminescence using Clarity Western ECL substrate (Biorad) and the FluorChemFC2 device with AlphaView software (Cell Biosciences).
Alphaview software
Manufactured by Cell Biosciences
Sourced in United States
AlphaView is a software application developed by Cell Biosciences for the analysis and quantification of data from various imaging and detection technologies. The core function of AlphaView is to provide a platform for users to process, analyze, and visualize data from their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Fluorchemfc2 device, by Cell Biosciences (2 mentions)
Mops buffer, by Bio-Rad (2 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (2 mentions)
Ultra sensitive enhanced chemiluminescent substrate kit, by 4A Biotech (2 mentions)
Enhanced bca protein assay kit, by Beyotime (2 mentions)
Fluorchem q imaging system, by Cell Biosciences (2 mentions)
Complete protease phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Bullet blender, by Next Advance (2 mentions)
Goat anti actin hrp, by Santa Cruz Biotechnology (1 mentions)
Criterion xt gel, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Protease inhibitors cocktail, by Roche (1 mentions)
Ab9484, by Abcam (1 mentions)
Criterion xt, by Bio-Rad (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti rabbit antibody, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Solarbio (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
10 protocols using alphaview software
1
Western Blot Analysis of Rat Colonocytes
2
Western Blot Analysis of Tight Junction Proteins
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Frozen colonic tissue was homogenized in a lysis buffer [15 (link)], and 25 µg of total protein lysates were loaded onto 4%–12% Criterion XT gel (Bio-Rad, Marnes-La-Coquette, France) before electrophoresis in MOPS buffer (Bio-Rad, Marnes-La-Coquette, France). After transfer onto nitrocellulose membrane and incubation in blocking solution (TBS pH 7.5, 0.05% Tween 20, and 5% (wt/vol) non-fat dry milk), membranes were incubated overnight (4 °C) with rabbit ZO-1 antibody (1/250, 617300, Invitrogen, Cergy Pontoise, France) or with mouse claudin-1 antibody (1/250, 374900, Thermo-Fisher Scientific, Bedford, MA, USA) diluted in blocking solution. After three washes, blots were incubated for 2 h at room temperature with an anti-rabbit or anti-mouse HRP-linked secondary antibody. Revealing was performed using enhanced chemiluminescence (ECL system, Pierce Biotechnology, Courtabœuf, France), and bands were quantified by densitometry using the FluorChem FC2 device and the AlphaView software (Cell Biosciences, Santa Clara, CA, USA). GAPDH expression (Ab9484, mouse monoclonal antibody, Abcam, Cambridge, UK) was used to ensure consistent protein loading and transferring.
3
Ovarian Protein Extraction and Western Blot Analysis
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RIPA Buffer was used to extract total proteins from ovaries. The BCA Protein Assay Kit was employed to detect the concentration of protein. Equivalent amounts of total protein were subjected to 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to PVDF membranes. After blocking with skim milk, the membranes were incubated overnight with an anti-GAPDH, anti-ASK1, anti-p-ASK1, anti-JNK, anti-p-JNK, anti-Bcl-2, anti-Bax, anti-caspase-9/3, anti-Cyt-c, anti-OPA1, anti-Mfn1, anti-Mfn2, anti-Drp1, anti-Fis1, or anti-PGC1a antibody (Table 2
4
Western Blot Analysis of Protein Expression
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For protein extraction, after the corresponding treatments, HCT-116 and HCT-15 cells, as well as tumor tissues, were washed twice with pre-cooled PBS and lysed on ice using RIPA lysis buffer containing 1 mM PMSF. Cell lysates were quantified using an Enhanced BCA Protein Assay Kit (Beyotime Biotechnology) according to the manufacturer’s protocol. Subsequently, equal amounts of proteins were separated by 10% SDS–PAGE and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking with 5% nonfat milk in PBST, the membranes were incubated with diluted specific primary antibodies at 4°C overnight, followed by incubation with the corresponding rabbit or mouse IgG horseradish peroxidase-conjugated secondary antibodies (diluted 1:5,000). Protein bands were detected with an Ultra-sensitive Enhanced Chemiluminescent Substrate Kit (4A Biotech) and visualized using a FluorChem E System (ProteinSimple, San Jose, CA, USA). The protein expression level was detected using AlphaView software (Cell Biosciences, Santa Clara, CA, USA).
5
Western Blot Analysis of Protein Expression
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Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing 1% phenylmethylsulfonyl fluoride (Solarbio) for 30 min on ice and the lysate was collected by centrifugation (15,000 × g for 10 min at 4°C). Protein concentration was quantified using an Enhanced BCA Protein Assay Kit (Beyotime Biotechnology). Equal amounts of proteins were loaded and separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States) that were incubated with the indicated primary antibodies followed by horseradish peroxidase-conjugated anti-rabbit or -mouse secondary antibody. The bands of interest were detected using an Ultra-sensitive Enhanced Chemiluminescent Substrate Kit (Beijing 4A Biotech Co., Ltd). β-tubulin was used as the loading control. Relative protein expression was quantified using AlphaView software (Cell Biosciences, Santa Clara, CA, United States). As for animal skin tissues, the protein lysates were performed for western blotting.
6
Myocardial Protein Expression Analysis
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Total proteins were drawn from myocardial tissues by using a RIPA lysis buffer. Protein concentration was measured using the BCA protein assay kit. An equal amount of total protein was dominated to sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (8%–12%), and distracting the proteins to PVDF membrane. A skim milk solution was used as a blocking agent. The membranes were incubated overnight, separately, with anti‐GAPDH, anti‐PGC‐1α, anti‐TFam, anti‐Mfn1/2, anti‐Drp1, anti‐Nrf1, anti‐PI3K, anti‐p‐Akt, anti‐Akt, anti‐p‐Bad, anti‐Bad, anti‐Bcl‐2, anti‐Bax, anti‐cleaved caspase‐9/3, anti‐caspase‐9/3, anti‐Cyt‐c and anti‐PARP antibodies (Table 2 ). Subsequently, the membrane was incubated with secondary HRP‐conjugated goat antibodies (anti‐rabbit) (Santa Cruz Biotechnology). The target proteins (Bax [green]/Bcl‐2 [red]) in the myocardial tissues were visualized using the enhanced chemiluminescence kit (Thermo Fisher Scientific). The target proteins were then subjected to densitometry using the Alpha View software (Cell Biosciences).
7
Western Blot Analysis of Protein Samples
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Proteins were extracted in RIPA lysis buffer (Solarbio) containing 1 mM PMSF (Solarbio). Samples containing equal amounts of protein were separated by SDS–PAGE and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore; Merck KGaA, Darmstadt, Germany). The membranes were probed with primary antibodies overnight at 4°C, and then incubated with the corresponding anti-rabbit or anti-mouse secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature. The bands of interest were detected using an Ultra-sensitive Enhanced Chemiluminescent Substrate Kit (4A Biotech, Co. Ltd., Beijing, China) and a FluorChem E System (ProteinSimple), and were further quantified using AlphaView software (Cell Biosciences, Santa Clara, CA, United States).
8
Quantitative Protein Analysis of Frozen Brains
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Frozen brain samples were cryopulverized, and the tissue was homogenized using glass beads and the Bullet Blender (NextAdvance) in ice-cold Tris-buffered saline buffer with 1% complete protease/phosphatase inhibitor (Thermo Scientific) and 2 mmol/L EDTA. Lysates were then centrifuged at 16,000 × g for 30 minutes at 4°C to pellet insoluble proteins. Supernatants were removed to clean tubes, and protein levels were measured using the BCA protein assay kit (Pierce, Rockford IL). Western blot analyses were performed as previously described (27 (link)); digital images were captured using the FluorChem Q imaging system (Cell Biosciences, Santa Clara, CA). Quantitative analysis was performed using Alpha View software (Cell Biosciences). Individual densitometry values were normalized to actin.
9
Protein Extraction and Western Blot Analysis
10
Quantitative Western Blot Analysis
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