Total protein was extracted using the RIPA buffer (Thermo Fisher Scientific, Inc.) from tissues and cells. The protein concentration was determined using a Pierce™ Rapid Gold BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of total protein (30 µg/lane) was separated using SDS-PAGE (10% gels) then, transferred onto PVD membranes (Merck KGaA). Subsequently, the membranes were blocked with 5% skimmed milk for 3 h at 4°C. After being washed three times with PBS, the membranes were incubated with the primary antibodies against SMO (1:1,000; cat. no. ab235183) or β-actin (1:2,500; cat. no. ab8226) (both from Abcam) overnight at 4°C. The secondary antibody Goat Anti-Rabbit IgG H&L [horseradish peroxidase (HRP)] (1:3,000; cat. no. ab205718; Abcam) and Goat Anti-Mouse IgG H&L (HRP) (1:10,000; cat. no. ab205718; Abcam) were added for incubation for another 2 h at room temperature after being washed three times with PBS at room temperature. The protein bands were visualized using Super ECL Detection Reagent (Vazyme Biotech Co., Ltd.) and the ChemiDoc™ MP Imaging System (Bio-Rad Laboratories, Inc.). ImageJ software V1.8.0_172 (National Institutes of Health) was used for semi-quantification of western blotting results.
Pvd membrane
Manufactured by Merck Group
Sourced in United States
PVD membranes are thin film coatings produced using physical vapor deposition (PVD) technology. They are designed to provide specialized surface properties and functional characteristics for various applications.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Pierce rapid gold bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Goat anti mouse igg h l hrp, by Abcam (1 mentions)
Alexa fluor 568 fluorescent dye conjugate, by Thermo Fisher Scientific (1 mentions)
Typhoon fla 9410 laser scanner, by GE Healthcare (1 mentions)
Goat anti mouse serum albumin antibody, by Fortis Life Sciences (1 mentions)
Monoclonal anti human factor 9 antibody, by Prolytix (1 mentions)
Chemiluminescence detection kit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ecl western blotting substrate kit, by Abcam (1 mentions)
Sftpc, by Abcam (1 mentions)
Sftpd, by Novus Biologicals (1 mentions)
β actin, by Abcam (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Applygen (1 mentions)
Enhanced chemiluminescence detection kit, by Zhongshan Biotechnology (1 mentions)
Ecl kit, by Merck Group (1 mentions)
9 protocols using pvd membrane
1
Western Blot Analysis of SMO Protein
2
Quantitative Western Blot Analysis of hFIX
3
Western Blot Analysis of CDK6 Protein
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Proteins were extracted using RIPA lysis buffer (Thermo Fisher Scientific, Inc.). BCA protein assay kit (Beyotime Institute of Biotechnology) was used to determine the protein concentration. Proteins (40 µg) were separated by 10% SDS-PAGE and transferred onto PVD membranes (EMD Millipore). Subsequently, the membranes were blocked in 5% non-fat milk and incubated with primary antibodies against CDK6 (1:1,000; cat. no. 3136S; Cell Signaling Technology, Inc.) and GAPDH (1:500; cat. no. ab8245; Abcam) overnight at 4°C. The following day, the membranes were blocked with appropriate HRP-conjugated secondary antibodies (1:1,000; cat. no. 7076S; Cell Signaling Technology, Inc.) at 37°C for 1 h. Lastly, the bands were visualized using a chemiluminescence detection kit (Thermo Fisher Scientific, Inc.). ImageJ 1 software (National Institutes of Health) was used for the quantification of band densities. GAPDH protein was used as the internal reference.
4
Western Blotting Analysis of Surfactant Proteins
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Western blotting analysis was carried out as described [51 (link), 58 (link), 59 (link)]. Briefly, exponentially growing imPAC2 cells were seeded in 60 mm dishes and cultured for 48 h. The supernatants were collected and dissolved in RIPA lysis buffer containing protease inhibitors, while the cells were directly lysed with RIPA lysis buffer containing protease inhibitors. The samples were subjected to SDS-PAGE and transferred to PVD membranes (Millipore). The membranes were blocked in 5% fat-free skimmed milk for 1 h, then incubated with antibodies against SftpA, SftpB, SftpD (Novus Biologicals, Shanghai, China), SftpC and β-actin (Abcam, Shanghai, China, 1:500) at 4 °C overnight, and subsequently incubated with respective secondary antibodies. The proteins of interest were visualized by using the ECL Western Blotting Substrate Kit (Abcam, Shanghai, China). β-actin (from cell lysate) was used as an internal control for both cell lysate and supernatant samples.
5
Western Blot Analysis of Protein Expression
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The testes or cells were lysed with a lysis buffer (Applygen Technologies Inc., Beijing, China), and the protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). The proteins (20 μg/well) per well were separated on 10% SDS-PAGE gel and then electrotransferred onto PVD membranes (Millipore, Bedford, MA, USA). The membranes were blocked with Tris-buffered saline (TBS, pH 7.4) containing 5% non-fat milk at room temperature for 1 h and then incubated with the primary antibodies overnight at 4 °C. The membranes were washed twice with TBS containing 0.1% Tween-20 and then incubated with the horseradish-peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Ag/Ab complexes were visualized using an enhanced chemiluminescence detection kit (Zhongshan Biotechnology Co.). β-Actin was used as the loading control. Band densitometry was quantified using ImageJ software (http://rsb.info.nih.gov/ij ).
6
Western Blotting Protocol for Protein Analysis
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Western blot analysis was carried as documented previously (4 (link)). Concisely, the RIPA buffer was employed to isolate proteins and quantified via the BCA assay. Equal concentrations of proteins were separated on a SDS-PAGE, then transfer-embedded onto PVD membranes (Millipore). After that, 5% non-fatty milk was employed to block the membranes, and inoculated with specified primary antibodies, namely Calnexin, HSP70, TSG101, Alix, and CD63, as described by the manufacturer. Membranes were then rinsed with the Dilute Tris Buffered Saline with Tween 20 (TBST-1x) solution, inoculated with specified peroxidase-labelled secondary antibodies and exposed with ECL kit (Millipore). All antibodies were obtained from Wuhan Sanying Biotechnology Co., Ltd (Wuhan, China).
7
Protein Lysate Extraction and Western Blotting
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Protein lysate was prepared from MIVs and ADSCs using the protein extraction reagent (Applygen, Beijing, China) according to the manufacturer's instructions. The protein lysates were subjected to SDS-PAGE, followed by being transferred onto a polyvinylidene difluoride (PVD) membranes (Millipore, Bedford, MA, USA). The membranes were blocked and then incubated with anti-Alix or β-actin antibody (1:500 dilution; Abcam) at 4 °C overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h followed by enhanced chemiluminescence (Thermo Fisher).
8
Western Blot Protein Detection
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Cells were lysed using radio-immunoprecipitation assay (RIPA) Lysis and Extraction Buffer (ThermoFisher, MT, USA). Lysate was loaded into 12% SDS-PAGE and transferred onto polyvinylidene difluoride (PVD) membrane (MilliPore, Darmstadt, Germany). After blocking with bovine serum albumin (BSA) in tris-buffered saline (TBS) with Tween20, membrane was incubated at 4°C overnight (1200 rpm) with corresponding primary antibodies according to the manufacturer's instructions. HRP-labeled secondary antibody was added and detected using Amersham™ ECL™ Start Western blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK). Protein bands were visualized by an image analyzer (LAS-4000 mid; GE healthcare).
9
Analyzing PLEKHA7 Expression in KG-1a Cells
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KG-1a cells were treated with Lipo-PLEKHA7-PEG-cRGD for 3 d, and further cultured for 2 d before lysis. The cells were suspended in a radioimmunoprecipitation (RIPA) lysis buffer (Santa Cruz Biotechnology, Dallas, TX, USA) for whole-cell lysis. Proteins were separated by SDS-PAGE and blotted onto the PVD membrane (Millipore). The same protocol was repeated with untreated KG-1a cells as a control cells to investigate PLEKHA7 expression in the cells before and after the addition of Lipo-PLEKHA7-PEG-cRGD. Images were captured, and chemiluminescent signals were analysed using ImageQuant LAS 4010 (GE Healthcare, Chicago, IL, USA). Western blot experiments were performed using the following antibodies: anti-PLEKHA7 (PA585686, 1:1000 working dilution, overnight shaking incubation at 4 °C), Cruz Marker molecular weight standards (sc-2035), β-actin (sc-47,778, 1:5000 working dilution, overnight shaking incubation at 4 °C) from Santa Cruz Biotechnology and secondary horseradish peroxidase (HRP)-conjugated antibody from Abcam (ab205718, 1:2000 working dilution, incubation at 25 °C for 1 h). Image J software was used to quantify PLEKHA7 protein expression in KG-1a cells.
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