The tissue samples were collected from humans and mice for Western blot analysis using a previously described protocol4 (link),25 (link). Briefly, total proteins were extracted according to the manufacturer’s protocol (Keygen Biotech, Nanjing, China). SDS-PAGE gels (5% stacking gel; 10% separating gel) were used to separate total protein lysates (40 μg per lane) and electrophoretically transferred to polyvinylidene fluoride membranes (PVDF) (Millipore Corporation, USA). PVDF membranes were incubated with 5% nonfat milk for 1 h at room temperature (RT) to prevent non-specific binding. Later, the membranes were incubated with rabbit anti-GHRH (1:500, catalog No: ab187512, Abcam, Cambridge, MA, USA) and anti-GAPDH (1:3,000, Proteintech, Wuhan, China) antibodies overnight at 4 °C. The secondary antibody (peroxidase-conjugated goat anti-rabbit IgG (1:3,000, Proteintech, Wuhan, China) was incubated with the PVDF membranes for 1 h at RT on the next day. Enhanced chemiluminescence (ECL) reagent (Thermo, Marina, CA, USA) and a Fusion FX5 image analysis system (Vilber Lourmat, Marne-la-Vallée, France) were used to visualize the bands. Finally, Quantity One software (Bio-Rad, CA, USA) was used to measure the resulting optical density (OD) values, which were normalized to GAPDH expression.
Polyvinylidene fluoride (pvdf)
Manufactured by Proteintech
Sourced in United States, China
PVDF (Polyvinylidene Fluoride) is a type of lab equipment used in various scientific applications. It is a durable and chemically resistant material that is commonly used for membrane filtration, Western blotting, and other analytical techniques. PVDF membranes are known for their high protein-binding capacity and the ability to efficiently transfer and immobilize proteins for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride (pvdf), by Merck Group (8 mentions)
Enhanced chemiluminescence ecl reagent, by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Fusion fx5 image analysis system, by Vilber (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Proteintech (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Enhanced chemiluminescence kit, by CWBIO (1 mentions)
Ripa lysis buffer, by CWBIO (1 mentions)
Mouse anti α tubulin, by Proteintech (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
β actin, by Proteintech (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
β tubulin, by Proteintech (1 mentions)
Polyvinylidene fluoride (pvdf), by Roche (1 mentions)
Western blotting luminol reagent, by Merck Group (1 mentions)
Foxo1, by Abcam (1 mentions)
Tgf β, by Abcam (1 mentions)
Stat3, by Abcam (1 mentions)
C myc, by Abcam (1 mentions)
9 protocols using polyvinylidene fluoride (pvdf)
1
Western Blot Analysis of GHRH and GAPDH
2
Protein Expression Analysis by Western Blot
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Cells transfected with siRNA for 48 hours were collected and lysed using RIPA Lysis Buffer (CWBIO) at 4°C for protein extraction; 20 µg of protein of each sample was electrophoresed by SDS-PAGE gel and then electrotransferred onto a polyvinylidene fluoride membrane (PVDF; Millipore, Billerica, MA, USA). Then 5% non-fat milk was used to block the PVDF membrane, followed by the incubation with primary antibodies (1:1000, Proteintech Group, IL, USA) at 4°C overnight. After incubation with secondary antibodies (1:3000, Proteintech Group) in blocking buffer, the bands were visualized using an enhanced chemiluminescence kit (CWBIO).
3
Western Blot Analysis of AKT3 Protein
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Protein lysates obtained from the cells were subjected to SDS-PAGE and then the gel was transferred to Polyvinylidene difluoride (PVDF, Merck Millipore). The PVDF membranes were blocked with 5% non-fat dry milk and then the membranes were incubated with rabbit anti-AKT3 (1:200, Proteintech, USA) or mouse anti-α-tubulin (1:2000, Proteintech, USA) overnight at 4 °C. The next day, they were incubated with the appropriate secondary antibodies (HRP-conjugated anti-rabbit IgG (1:5000, CST, USA) or HRP-conjugated anti-mouse IgG (1:5000, CST,USA) and detected with a chemiluminescence imaging analysis system (Tanon, China).
4
Western Blot Analysis of HELLS Protein
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Cells were collected and lysed in cold RIPA buffer supplemented with phenylmethanesulfonyl fluoride (PMSF; Beyotime, China) and centrifuged at 14,000 rpm and 4 °C for 15 min. Total protein concentrations were determined with a BCA protein assay kit (Beyotime, China). Equal amounts of denatured protein samples were separated by electrophoresis using 10% SDS-PAGE gel fast preparation kits (Epizyme, China), and then transferred onto 0.45 μm polyvinylidene difluoride (PVDF; Millipore, USA) membranes. Subsequently, the membranes were blocked with 5% BSA diluted in Tris-buffered saline with 0.1% Tween-20 (TBST) at room temperature. Then, the PVDF membranes were incubated with primary antibodies against β-actin (1:1000; Proteintech) and HELLS (1:1000; Proteintech) at 4 °C overnight. The next day, the primary antibody was washed three times with TBST and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h.
5
Western Blot Analysis of PDHB
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RIPA whole-cell lysis solution was used to lyse the cells. Total proteins were then extracted, measured, and semi-dry transferred to PVDF (Millipore, Billerica, MA, USA) membranes after separation by 8–12% SDS-PAGE electrophoresis; PVDF membranes were first blocked with TBS + Tween (TBST) solution containing 5% skim milk for 2 h. Then they were washed, and incubated with primary antibody at 4 ◦C overnight (PDHB, 12649-1-AP, Proteintech, 1:50); they were then rewashed and incubated for 2 h with a secondary antibody that was colored by horseradish peroxidase. The PVDF membrane was washed before the chemiluminescent substrate was applied and then developed by a gel imaging system, after which the grayscale values were measured. The membranes were trimmed reasonably before chemiluminescence (the original length is preserved).
6
FSTL1 Protein Expression Analysis
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Briefly, the total protein from samples was separated by electrophoresis (10% SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane (PVDF) (Roche, Switzerland). Then, we incubated PVDF membranes with primary antibodies against FSTL1 (1 : 2000, ProteinTech, USA) and β-Tubulin (1 : 5000, ProteinTech, USA) at 4°C overnight, followed by washing with TBST and secondary antibodies at room temperature for one hour. Finally, the protein visualization was achieved by an enhanced chemiluminescence (Thermo Fisher Scientific, USA) detection system.
7
Western Blot Analysis of TCIRG1 Protein
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RIPA whole-cell lysis solution was used to lyse the cells. Total proteins were then extracted, measured, and semi-dry transferred to PVDF (Millipore, Billerica, MA, USA) membranes after separation by 8–12% SDS-PAGE electrophoresis; PVDF membranes were first closed with TBS + Tween (TBST) solution containing 5% skim milk powder for 2 h, washed, and incubated with primary antibody at 4 °C overnight (TCIRG1, 12649-1-AP, Proteintech, 1:50); they were then rewashed and incubated for 2 h with a secondary antibody that was colored by horseradish peroxidase. The PVDF membrane was washed before the chemiluminescent substrate was applied and then developed by a gel imaging system, after which the grayscale values were measured.
8
Quantification of Cellular Proteins
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Total proteins from cells and tissues were obtained with RIPA buffer (Thermo Fisher Scienti c) containing protease inhibitor cocktail. Then, the protein concentrations were determined by using the BCA Protein Assay Kit (Thermo Fisher Scienti c) based on the instructions. After that, equal amounts of protein were resolved using 10% SDS-PAGE gels and then transferred to the polyvinylidene uoride (PVDF; Millipore, Dallas, Tx, USA) membranes. The 5% skim milk was used to block the PVDF membranes, which was then incubated with the primary antibodies overnight at 4 °C, including TCEAL7 (cat no. 11218-1-AP, Proteintech, Wuhan, China), AKT1 (cat no. ab235958, Abcam, Cambridge, MA, USA), AKT2 (cat no. ab175354, Abcam), c-Myc (cat no. ab32072, Abcam), STAT3 (cat no. ab68153, Abcam), β-catenin (cat no. ab16051, Abcam), TGF-β (cat no. ab179695, Abcam), YAP (cat no. ab52771, Abcam), FOXO1 (cat no. ab52857, Abcam) and GAPDH (cat no. 60004-1-Ig, Proteintech). The membranes were then incubated with secondary antibodies for 1 hour at room temperature. Western blotting luminol reagent (Millipore) was used to visualize the immunoreactive bands.
9
Western Blot Analysis of HCF Proteins
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Total protein of HCFs with different treatments was extracted with RIPA buffer, and protein concentration was examined using a bicinchoninic acid protein assay kit (Beyotime Biotechnology, Jiangsu, China). Then, equal amounts of total protein from each group were separated by electrophoresis on 10% sodium dodecyl sulfatepolyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) (Merck, Darmstadt, Germany) membrane. The proteins on PVDF membrane were blotted with 5% nonfat milk and incubated respectively with rabbit polyclonal antibodies of α-SMA, ALDH2, or periostin (1:1000; # 55135-1-AP, # 15310-1-AP, # 19899-1-AP, Proteintech Group, Wuhan, China), or rabbit monoclonal antibodies of Smad2, Smad3, phospho-Smad2, or phospho-Smad3 (1:1000; # 5339, # 9523, # 18338, # 9520; Cell Signaling Technology, Danvers, MA) overnight. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (1:10,000; # 60004-1-lg, Proteintech Group) was adopted as an internal control. Finally, the PVDF membranes were washed, incubated with secondary antibodies labeled with horse radish peroxidase, and visualized by the enhanced chemiluminescence reagents (Merck). The intensity of protein bands was calculated by the Image-Pro Plus 6.0 software. Each experiment was performed for 3 times.
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