Samples were diluted to at least 1:5 with sample buffer, heated at 95 °C for 5 min, and then stored at 4 °C until they were used. The gel was run at 80 V for 10 min and then at 130 V for 3 h. Polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Burlington, MA, USA) were soaked in methanol for 1 min and then placed in the “sandwich” chamber with 2 fiber pads and 2 filter papers, all soaking in old transfer buffer. The “sandwich” was transferred for 1.5 h at 100 V at 4 °C. The membranes were then shaken in 5% nonfat dry milk in PBS for 1 h on a shaker at room temperature; they were then incubated with a primary anti-KCNQ2 antibody (1:200) (Thermo Fisher, Waltham, MA, USA) in 1% milk at 4 °C on a shaker overnight. The next day, after it had been washed with PBST (phosphate buffer saline + Tween 20) 4 times for 10 min each time, the membrane was incubated with a secondary antibody (anti-rabbit) (1:3,000) (Gentex, Carbondale, PA, USA) in 1% milk prepared with PBS for ~ 1 h at room temperature, rinsed with PBST 4 times for 10 min each time, and then analysed using a western blotting detection kit (Advansta, Menlo Park, CA, USA). Anti-glyceraldehyde phosphate dehydrogenase (GAPDH) antibody was used as a loading control.
Western blotting detection kit
Manufactured by Advansta
Sourced in United States
The Western blotting detection kit is a laboratory equipment designed for the detection and visualization of proteins separated by gel electrophoresis. The kit contains the necessary reagents and materials for the chemiluminescent detection of target proteins on a membrane after the Western blotting process.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibodies, by Abbkine (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Bca protein concentration assay kit, by Biosharp (1 mentions)
Anti β actin, by Bioss Antibodies (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
P1260, by Applygen (1 mentions)
Goat anti rabbit igg, by Absin (1 mentions)
Rabbit anti p erk1 2, by Cell Signaling Technology (1 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
Rabbit anti p p38, by Cell Signaling Technology (1 mentions)
Rabbit anti p akt, by Cell Signaling Technology (1 mentions)
Rabbit anti pstat3, by Cell Signaling Technology (1 mentions)
Chemidoc xrs image analyser, by Bio-Rad (1 mentions)
7 protocols using western blotting detection kit
1
Western Blot Protein Detection Protocol
2
Western Blot Analysis of Recombinant Proteins
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Total proteins were extracted from mycelia cultured in liquid CM for 48 h as previously described (Li et al., 2011 (link)). Proteins were separated with 10% SDS‐PAGE and then transferred to polyvinylidene fluoride (PDVF) membranes. The target proteins on the PDVF membrane were detected with anti‐GFP and anti‐RFP antibodies (Abbkine), along with horseradish peroxidase (HRP)‐conjugated secondary antibodies (Abbkine). Signals were visualized with a Western Blotting Detection Kit (Advansta) and the membrane was photographed using the ChemiDoc Touch imaging system (Bio‐Rad). The protein loading was monitored by staining proteins in acrylamide gels with Coomassie Brilliant blue (CBB) for 2 h and destaining the gel overnight before imaging (Wang et al., 2018 (link)).
3
Quantitative Western Blotting of Myometrial Proteins
4
Tissue-Specific Protein Expression Profiles
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Total protein was extracted from the liver, kidney, jejunum, and ileum (100 mg, n = 3 chickens for each group) using RIPA cell lysis buffer (BL504A, Biosharp, China) and protein phosphatase inhibitor (P1260, Applygen, China). Protein concentrations were determined using a BCA protein concentration assay kit (BL521A, Biosharp, China). Proteins were electrophoresed using an electrophoresis apparatus (EPS 300, Tanon, China). The following antibodies were used: anti-BCRP (cat. no. bs-0662R, polyclonal, 1:1000, Bioss, China), anti-MRP4 (cat. no. bs-1422R, polyclonal, 1:1000, Bioss, China), anti-β-actin (cat. No. abs137975, monoclonal, 1:1000, Absin, China), and goat anti-rabbit IgG (cat. no. AP132P, 1:1000, Millipore, USA). Immunocomplexes were visualized using a western blotting detection kit (Advansta, USA) and blots were imaged using a ChemiDoc MP Imaging System (Bio-Rad, USA). Band densities were analyzed by Image-Pro Plus 6.0 and were normalized to those of β-actin. The protein relative expression levels were normalized to the average level of the NC group.
5
Western Blot Analysis of Signaling Proteins
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The cell lysates were prepared using a radioimmunoprecipitation cell‐lysis buffer (Thermo Fisher Scientific). The total protein concentration was determined using the bicinchoninic acid reagent system (Thermo Fisher Scientific). The protein extracts (20 μg of total cell protein) were subjected to 7.5–15% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene difluoride membranes. Nonspecific binding was blocked using Tris‐buffered saline with 0.5% Tween‐20 containing 5% skimmed milk powder. Specific antigens were immunodetected using appropriate primary and secondary antibodies and visualized using a western blotting detection kit (Advansta, San Jose, CA). Membranes were stripped using Restore Western Blot Stripping Buffer (Thermo Fisher Scientific) and reprobed for GAPDH to confirm equal loading. The primary antibodies were: rabbit anti‐pSTAT3, rabbit anti‐pERK1/2, rabbit anti‐pSARK/JNK, rabbit anti‐pp38, rabbit anti‐pAKT and rabbit anti‐GAPDH (1:1,000; Cell Signaling Technology). The secondary antibodies were all obtained from Cell Signaling Technology (1:3,000).
6
Detecting YxaL Protein by Western Blot
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Purified YxaL was utilized as antigen to generate a rabbit polyclonal antibody for detecting YxaL by western blotting in subcellular compartments of strain GH1-13 during its growth in TSB medium. A purified anti-YxaL IgG was used as a primary antibody (dilution rate, 1:20,000), which was then detected using a secondary chicken anti-rabbit IgG antibody with horseradish peroxidase (HRP) conjugate (Abcam, Cambridge, UK) and a western blotting detection kit (Advansta, Menlo Park, USA). Chemiluminescent images were acquired using a ChemiDoc XRS image analyser (Bio-Rad, Hercules, USA), and images were manipulated using Molecular Dynamics ImageQuant version 5.2 (GE Healthcare, Waukesha, USA).
7
Western Blot Analysis of KCNQ2 Protein
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Samples were diluted to at least 1:5 with sample buffer, heated at 95 °C for 5 min, and then stored at 4 °C until use. The gel was run at 80 V for 10 min and then at 130 V for 3 h. To prepare for Western blotting, the polyvinylidene difluoride (PVDF) membrane (Millipore) was soaked in methanol for 1 min and then placed in the “sandwich” chamber with 2 fiber pads and 2 filter papers that absorbed the old transfer buffer. The “sandwich” was transferred for 1.5 h at 100 V at 4 °C. The membrane was then shaken in 5% nonfat dry milk in PBS for 1 h in a shaker at room temperature; it was then incubated overnight with a primary anti-KCNQ2 antibody (1:200; Thermo Fisher) in 1% milk at 4 °C in a shaker. The next day, after it had been washed with PBST (phosphate buffer saline + Tween20) four times for 10 min each time, the membrane was incubated with a secondary antibody (antirabbit) (1:3000; Gentex) in 1% milk prepared with PBS for approximately 1 h at room temperature. It was then rinsed with PBST four times for 10 min each time and analyzed using a Western blotting detection kit (Advansta, Menlo Park, CA, USA). An anti-GAPDH antibody was used as the internal control and anti-pan cadherin was used as a cell membrane marker.
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