The LMH cells infected with FAdV were collected and lysed in lysis buffer (CST, MA, USA) with PMSF (Beyotime, Shanghai, China), protease and phosphatase inhibitors (CST, MA, USA). The lysates were boiled in the loading buffer and were then subjected to 10% SDS-PAGE and transferred to nitrocellulose (NC) membranes (GE Healthcare Life sciences, Freiburg, Germany). After blocking with 5% skimmed milk in PBST for 1 h at room temperature (RT), the membranes were reacted with the corresponding antibodies at 4 ℃ overnight. After being washed with PBST three times, the membrane was incubated with HRP-labelled secondary antibodies for 1 h at RT. After another three washes, the membranes were developed with chemiluminescent reagents and imaged with an automatic imaging system (Tanon 5200).
Nc membrane
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Germany
The NC membrane is a versatile laboratory filter material made of nitrocellulose. It is commonly used in various applications where filtration, separation, or immobilization of biological or chemical samples is required. The membrane provides a high surface area and consistent pore size, making it suitable for a range of filtration and analysis tasks.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (6 mentions)
Sds page, by Beyotime (3 mentions)
Loading buffer, by Solarbio (2 mentions)
Np 40 lysis buffer, by Beyotime (2 mentions)
Protease inhibitor mixture, by Roche (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Nitrocellulose, by GE Healthcare (2 mentions)
Nc membranes, by Abcam (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ecl reagent, by Cytiva (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Gfp mouse monoclonal antibody, by Proteintech (2 mentions)
Gapdh mouse monoclonal antibody, by Proteintech (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemi luminescence (ecl), by Vazyme (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
45 protocols using nc membrane
1
Western Blot Analysis of FAdV Infection
2
Protein Immunoblotting with SDS-PAGE
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Protein with equal concentrations in each group were mixed with 5× protein loading buffer (Sigma-Aldrich, United States) and incubated at 99°C for 10 min. Then running the sample by 15% SDS-PAGE gels before being subsequently transferred to NC membranes (GE, United States). The immnunoblots were incubated with 5% milk at room temperature for 0.5–1 h, followed by an incubation at 4°C with primary antibodies overnight. After rinsing the membranes five times with TNET buffer (5 min of each), incubated with IgG (H + L)-HRP secondary antibodies (Santa Cruz, United States) for 1–2 h. The band densities were detected using an LumiBest ECL solution from Shanghai Sharebio Biotechnology (Shanghai, China) and Bio-Rad Laboratories (BIO-RAD, United States). ImageJ Software was using for analysis of the results.
3
Immunoblotting Analysis of HA Expression
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MDCK cells were infected with the H1-H16 subtype corresponding recombinant viruses or 293 T cells were transfected with pCAGGS-H17HA or pCAGGS-H18HA plasmid. Cells were collected and lysed with NP-40 lysis buffer (Beyotime, Shanghai) after 24 h’ infection or transfection. Cell lysates were mixed with 4 × loading buffer (Solarbio, Beijing) and denatured at 100 °C for 15 min, then were separated using SDS-PAGE with a 10% polyacrylamide gel [44 ] and transferred to NC membranes (GE Healthcare, Amersham). Next, membranes were blocked with 5% skimmed milk in PBS at 37 °C f for 1 h, then washed six times (5 min per time) with PBST and incubated with primary antibody 3C12 (1:800) at 4 °C overnight. Then, after six times washing, the membrane was incubated with HRP-conjugated goat anti-mouse antibody (1:10000; KPL, Gaithersburg, MD) as secondary antibody at 37 °C for 1 h. Membranes were then washed and the target protein bands were detected with enhanced chemiluminescence (ECL) (Vazyme, Nanjing) and the signals were recorded using Image Lab Software (Bio-Rad). GADPH served as a loading control.
4
Western Blot Analysis of Protein Lysates
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Total protein lysate concentration was determined using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA), whereby 30–50 μg of protein lysate was heated at 95°C in the sample buffer for 5 min. Denatured proteins were separated in SDS-PAGE with a running buffer consisting of 25 mM Tris-HCl, 192 mM glycine, and 0.1% SDS, before being transferred to polyvinylidene fluoride (PVDF) membranes (0.45 μM; Millipore, Merck KGaA, Darmstadt, Germany) or NC membranes (0.22 μM, Amersham, GE Healthcare Life Sciences, Pittsburgh, PA, USA) with transfer buffer (700 mL of ddH2O, 200 mL of methanol, and 100 mL of 10× running buffer [250 mM Tris-HCl and 1.92 M glycine]). The transferred membrane was blocked with 5% milk or BSA in Tris-buffered saline-Tween (10 mL of 2 μM Tris-HCl, pH 7.4, 100 mL of 5 M NaCl, 0.5 mL of 100% Tween 20, and 890 mL of ddH2O) and stained with the indicated primary antibodies at 4°C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies. ECL signaling was detected using a ChemiDoc Touch Imaging System (Bio-Rad).67
5
Western Blot Analysis of Cellular Proteins
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The LMH cells transfected with plasmids or infected with FAdV were collected and lysed in lysis buffer with PMSF, protease, and phosphatase inhibitors. The lysates were boiled in the loading buffer, and was then subjected to 10% SDS-PAGE and transferred to nitrocellulose (NC) membranes (GE Healthcare Life sciences, Freiburg, Germany). After blocked with 5% skim milk in PBST for 2 h at room temperature (RT), the membranes were reacted with the corresponding antibodies at 4°C overnight. After being washed with PBST for three times, the membrane was incubated with HRP-labeled secondary antibodies for 1 h at RT. After another three washes, the membranes were developed with chemiluminescent reagents and imaged with an automatic imaging system (Tanon 5200).
6
Western Blotting of Suffruticosol C Treated Cells
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The Western-blotting protocol was the same as outlined in our previous study [37 (link)]. Briefly, cells were plated in a 24-well plate at a density of 3 × 105 cells/well and reached 70−80% confluence before suffruticosol C treatment. After different concentrations of suffruticosol C treatments for 24 h, the collected cells were washed with PBS and suspended in a Radio Immune Precipitation Assay (RIPA) buffer on ice for 30 min. Proteins were separated using SDS-PAGE and transferred onto nitrocellulose (NC) membranes (0.45 µM, GE). The NC membrane was exposed to the primary antibody for an overnight incubation, followed by three PBS washes. Sequentially, the membranes were then exposed to a horseradish peroxidase (HRP)-conjugated secondary antibody for an incubation of 2 h at room temperature and washed with PBST three times. Finally, the proteins were detected and quantified using the Imaging System (Bio-Rad, Hercules, CA, USA) and ImageJ software (National Institutes of Health, Silver Springs, MD, USA), respectively.
7
Protein Extraction and Western Blot Analysis
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Total protein was extracted from treated cells or tumor tissues in RIPA lysis buffer and separated by 8% or 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) (Beyotime, China) before being transferred onto NC membranes (GE Healthcare, UK). The membranes were blocked in 5% skim milk for 1 hour at room temperature and then incubated with primary antibody overnight at 4 °C. Subsequently, the membranes were incubated with secondary antibody for 1 hour at room temperature. The immunoreactive bands were detected using Clarity TM Western ECL substrate (Bio-Rad, USA) and a Bio-Rad GelDoc system (Bio-Rad, USA). The primary antibodies and dilutions used are listed in Supplementary Table 11 .
8
Western Blot Analysis of RABV Infection
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SK-N-SH cells infected with RABV for 72 h were collected and lysed in NP-40 lysis buffer (Beyotime, Shanghai, China). Cell lysates were mixed with 4× loading buffer (Solarbio, Beijing, China) and boiled at 100 °C for 15 min. Protein samples and recombinant M protein samples were separated using SDS-PAGE with a 10% polyacrylamide gel [21 (link)] and transferred onto NC membranes (GE Healthcare, Amersham, UK). After blocking with 5% non-fat milk at 37 °C for 1 h, NC membranes were washed three times with PBST and incubated with the undiluted supernatant of hybridoma cells or mouse anti-His antibody (1:1000; Abcam, Cambridgeshire, UK) and anti-N mAb (produced in our laboratory) [22 (link)] as primary antibody at 4 °C overnight. Then, the membrane was incubated with HRP-conjugated goat anti-mouse antibody (1:10,000; KPL, Gaithersburg, MD, USA) as secondary antibody at 37 °C for 1 h. Membranes were then washed and the target protein bands were detected with enhanced chemiluminescence (ECL) (Vazyme, Nanjing, China).
9
Western Blot Analysis of DNA Repair Proteins
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Protein extracts, separated by SDS-PAGE and transferred onto NC membranes (General Electric Company, Schenectady, NY, USA), were probed with antibodies against XPF (D3G8C, Rabbit mAb, #13465), Rabbit anti-γ-H2AX Recombinant mAb BLR053F (A700-053, 1:1000, Bethyl, Gurgaon, India), Anti-RAD 51 antibody (NBP2-20058, 1:1000, Bio-Techne, Minneapolis, MN, USA), or Anti-beta Actin antibody (mAbcam 8226, 1:1000, Abcam, Cambridge, UK). Proteins of interest were detected with peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG, Fc Fragment Specific (115-035-008, 1:1000, Jackson ImmunoResearch, Bar Harbor, ME, USA), or goat anti-rabbit IgG, Fab’2-HRP (sc-3837, Santa Cruz Biotechnology, CA, USA) and visualized with the Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Merck, Rahway, NJ, USA), according to the protocol provided. The quantification of WB bands was achieved through Image J (ImageJ bundled with 64-bit Java 8. https://imagej.nih.gov/ij/(accessed on 28 June 2022)).
10
Protein Expression Analysis Protocol
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The total cell lysates were prepared by resuspending 0.45×106 cells in 20–50 µl of RIPA lysis buffer (50 mM Tris buffer, 20 mM HEPES, 100 mM NaF; 120 mM NaCl, 0.5% Triton X-100, 100 µM Na3VO4, pH 7.6) Total lysates was quantified using a BCA kit (#23225; Thermo Fisher Scientific) according to the manufacturer's protocols. The proteins (30–70 µg) were isolated using 10 or 12% SDS-PAGE gels and electrotransferred onto NC membranes (GE Healthcare). Target proteins were identified using the respective antibodies and Immobilon Western Chemiluminescent HRP Substrate Solution (WBKLS0100; Millipore) and visualized by Davinch-Chemi (CAS-400SM; Davinch-K). Anti-PARP antibody (1:1,000; #9542) and anti-Bax (1:1,000; #2772) were obtain from Cell Signaling Technology. Anti-caspase-3 (1:3,000; ADI-AAP-113) and anti-FLIP (1:700; ALX-804-961-0100) were purchased from Enzo Life Sciences. Anti-Bcl-2 (1:700; sc-7832), anti-Bcl-xL (1:1,000, sc-634), anti-Mcl-1 (1:1,000; sc-12756), anti-cIAP1 (1:1,000; sc-7943), anti-cIAP2 (1:1,000; sc-517317) and anti-β-actin antibody (1:5,000; sc-47778) were supplied by Santa Cruz Biotechnology, and anti-XIAP (1:10,000; 610717) antibody was obtained from BD Biosciences.
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