The Laemmli sample buffer was used in cell lysis to prepare whole cell lysates for SDS-PAGE and WB, as described previously [24 (link),28 (link),29 (link)]. The primary antibodies against Numb (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-136554), Myc tag (Santa Cruz Biotechnology, sc-40), GFP (Biolegend, San Diego, CA, USA, 75818-584), ubiquitin (Santa Cruz Biotechnology, sc-8017), GAPDH (Santa Cruz Biotechnology, sc-365062), TUBB1/β-tubulin (Sigma, Livonia, MI, USA, T7816), ZIKV C (GeneTex, Irvine, CA, USA, GTX133317), ZIKV E (B.E.I. Resources, Newport News, VA, USA, NR-50413), ZIKV NS4B (GeneTex, GTX133311), and ZIKV NS5 (GeneTex, GTX133329) were used in this study. Goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (Bio-Rad, Hercules, CA, USA, 170–5046, and 170–5047) were used as secondary antibodies in this study. For revealing the specific reactions, a chemiluminescence substrate was used, and the signal was recorded digitally using a Bio-Rad ChemiDoc XRS imaging system with the QuantityOne Program, version 4.6 (Bio-Rad Laboratories, CA). Densitometry analysis of the WB images was performed with the QuantityOne Program, version 4.6 (Bio-Rad). All WB images were acquired in the linear range of digital intensity without saturated pixels.
Quantity one program version 4
Manufactured by Bio-Rad
Sourced in United States, Germany
Quantity One is a software program developed by Bio-Rad for analyzing and quantifying data from gel electrophoresis and other experimental techniques. Version 4.6 is the latest release, providing tools for image capture, analysis, and data management.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xrs imaging system, by Bio-Rad (5 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Gel doc xr, by Bio-Rad (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Bca kit, by Beyotime (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Poly adp ribose polymerase parp, by Santa Cruz Biotechnology (1 mentions)
Heat shock protein 90 hsp90, by Santa Cruz Biotechnology (1 mentions)
Stat1, by Cell Signaling Technology (1 mentions)
Phospho stat1, by Cell Signaling Technology (1 mentions)
Goat anti mouse igg, by Boster Bio (1 mentions)
Westernbright sirius detection kit, by Advansta (1 mentions)
Hrp conjugated affinipure goat anti rabbit igg, by Boster Bio (1 mentions)
Goat anti mouse igg, by Rockland Immunochemicals (1 mentions)
β tubulin, by Merck Group (1 mentions)
Anti tubulin antibody, by Santa Cruz Biotechnology (1 mentions)
11 protocols using quantity one program version 4
1
Immunoblotting of Zika Virus Proteins
2
Western Blot Analysis of GFP and GAPDH
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To verify the expression of GFP and GAPDH, GFP-positive PK15 cells were collected and lysed in RIPA buffer (Sigma-Aldrich, St. Louis, CA, USA), and the total proteins were quantified with a BCA kit (Beyotime, Shanghai, China). An equal amount of the total proteins from each sample was separated on 12% sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany). Then, membranes were blocked with 5% non-fat milk and incubated with GAPDH and GFP antibody at dilution of 1:1000 (Proteintech, Rosemont, IL, USA). After washing with TBST, HRP-conjugated anti-Rabbit IgG was used as the secondary antibody. Digital signal of chemiluminescent Western blotting was acquired by Bio-Rad GelDoc XR and ChemiDoc XRS system, and analysis was conducted by the Quantity One program, version 4.6 (Bio-Rad, California, CA, USA).
3
Western Blot Analysis of Protein Expression
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HeLa or PK-15 cells were lysed in the precooled lysis buffer added with phenylmethylsulfonyl fluoride (PMSF; Beyotime, China). The samples were resolved in a 12% sodium dodecyl sulfate-polyacrylamide gel. Separated proteins were then transferred onto a nitrocellulose membrane and incubated with antibody against STAT1 (Cell Signaling, Danvers, MA, USA), phospho-STAT1 (Tyr701, herein named STAT1-Y701, Cell Signaling), FLAG (Sigma-Aldrich, St. Louis, MO, USA), HA (Sigma-Aldrich), heat shock protein 90 (HSP90; Santa Cruz Biotechnology, Santa Cruz, CA, USA), poly (ADP-ribose) polymerase (PARP; Santa Cruz Biotechnology), rabbit anti-3C serum (kept in our laboratory) or β-actin antibody (Santa Cruz Biotechnology), respectively. The membranes were then incubated with HRP-conjugated affinipure goat anti-rabbit IgG or goat anti-mouse IgG (Boster, Wuhan, China), respectively. The membranes were then developed using WesternBright™ Sirius detection kit on the basis of the manufacturer's instructions (Advansta, Menlo Park, CA, USA). Digital signal was acquired and analyzed by the Quantity One program, version 4.6 (Bio-Rad).
4
Protein Expression and Western Blot Analysis
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Cells were lysed in Laemmli sample buffer. The lysate samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, as previously described [17 (link)]. Primary antibodies against green fluorescence protein (GFP) (Santa Cruz Biotechnology, Inc., Dallas, TX), IRF3 and phosphorylated IRF3-S396 (Cell Signaling Technology, Danvers, MA), HA (Thermo Fisher), Myc (Thermo Fisher), FLAG (Sigma-Aldrich, St. Loius, MO), GAPDH (Santa Cruz), TBK1 and phosphorylated TBK1 (Cell Signaling), IRF3 (Santa Cruz), MAVS (Santa Cruz), and β-tubulin (Sigma) were used in the blotting. Horseradish peroxidase-conjugated secondary antibodies used in this study were goat anti-rabbit or goat anti-mouse IgG (Rockland Immunochemicals). The chemiluminescence signal was collected and analyzed digitally using a ChemiDoc XRS imaging system (Bio-Rad Laboratories, Hercules, CA) and the Quantity One Program, version 4.6 (Bio-Rad).
5
PTEN Protein Detection by Western Blot
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The SDS-PAGE and Western blot analyses were conducted as previously described [11 (link),13 (link)]. Briefly, after denatured proteins were transferred to a PVDF membrane, the membrane was blocked and probed by rabbit anti-PTEN antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Specific reactions between different antibodies and corresponding proteins were detected by using goat anti-rabbit conjugated with horseradish peroxidase (Sigma, St. Louis, MO) and revealed by a chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA). The membrane was also probed with anti-Tubulin antibody (Santa Cruz) to normalize the total protein loading. The chemiluminescence signal was digitally recorded and analyzed by the ChemiDoc XRS imaging system (Bio-Rad, Hercules, CA) with Quantity One Program (Version 4.6, Bio-Rad).
6
Western Blot Analysis of sRNase L Protein
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PK-15 and sRNase L KO-PK cells were grown in 6-well plates for 16 h and lysed in ice-cold cell lysis buffer supplemented with phenylmethylsulfonyl fluoride (PMSF; Beyotime, Shanghai, China). Samples of cell lysates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Briefly, the samples were resolved in a 12% polyacrylamide gel. Separated proteins were then transferred onto a nitrocellulose membrane and probed with anti-sRNase L antibody or β-actin antibody (Solarbio, Beijing, China). Specific reaction products were detected with horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Boster, Wuhan, China). The membranes were developed using SuperSignal West Pico Chemiluminescent Substrate according to the manufacturer's suggestions (Pierce, Rockford, IL, USA). Digital signal acquisition and analysis were conducted by the Quantity One program, version 4.6 (Bio-Rad).
7
Western Blotting for PRRSV and Swine IL-2
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Western blotting was carried out as described previously [10] (link). Briefly, the cell lysates were resolved in a 10% polyacrylamide gel. The separated proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and probed with PRRSV-specific antiserum of pigs or mouse monoclonal antibody against swine IL-2 (Invitrogen, Camarillo, CA, USA), respectively. Specific reaction products were detected with horseradish peroxidase-conjugated Staphylococcal Protein A (SPA-HRP, Boshide, Wuhan, China) or HRP-conjugated goat anti-mouse IgG (Boshide, Wuhan, China). The membranes were developed using Supersignal West Pico Chemiluminescent Substrate according to the manufacturer's suggestions (Pierce, Rockford, IL, USA). Digital signal acquisition and analysis were conducted by the Quantity One program, version 4.6 (Bio-Rad).
8
Western Blot Analysis of Protein Expression
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Cell lysates were prepared from infected or uninfected cells at indicated time points post infection (p.i.) with the Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime) supplemented with proteases and phosphatase inhibitors (Roche, Basel, Switzerland). Protein samples were electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA, United States) and probed with primary antibodies, which were further bound to HRP-conjugated secondary antibodies after washes. Finally, the PVDF membrane was immersed by Immun-Star™ HRP Substrate (Bio-Rad, Hercules, CA, United States) and images were captured using a ChemiDoc™ XRS + imaging system (Bio-Rad). Digital signal acquisition and analysis were conducted using the Quantity One program, version 4.6 (Bio-Rad).
9
Lung Protein Isolation and Western Blot
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Total protein was isolated from the right lung by homogenization with a buffer as previously described [14 (link)]. The samples were clarified by centrifugation at 14000 g for 5 min to remove the debris and intact cell. Then the supernatant was lysed in Laemmli sample buffer. The whole proteins in the lysate were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot as described previously [15 (link), 16 (link)]. Antibodies against Smad2/3 (Santa Cruz Biotechnology, Santa Cruz, CA), Smad7 (Santa Cruz Biotechnology), and actin (Sigma-Aldrich, St. Louis, MO) were used in the blotting. The chemiluminescence signal was recorded digitally using a ChemiDoc XRS imaging system (Bio-Rad Laboratories, Hercules, CA). Digital signal acquisition and densitometry analyses were conducted using the Quantity One Program, Version 4.6 (Bio-Rad).
10
Quantifying pCD163 Protein Expression
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After being grown in 6-well tissue culture plates for 48 h, MARC-145 and pCD163-MARC cells were lysed in ice-cold cell lysis buffer (Beyotime, Shanghai, China) containing 20 mM Tris (pH 7.5), 150 mM NaCl, and 1% Triton X-100 supplemented with phenylmethylsulfonyl fluoride (PMSF; Beyotime, Shanghai, China). The samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Briefly, the samples were resolved in a 12% polyacrylamide gel. Separated proteins were then transferred onto a nitrocellulose membrane and probed with mouse anti-pCD163 MAb or β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Specific reaction products were detected with horseradish peroxidase- (HRP-) conjugated goat anti-mouse IgG (Boster, Wuhan, China). The membranes were developed using SuperSignal® West Pico Chemiluminescent Substrate according to the manufacturer's suggestions (Pierce, Rockford, IL, USA). Digital signal of chemiluminescent Western blotting was acquired by Bio-Rad GelDoc™ XR & ChemiDoc™ XRS system and analysis was conducted by the Quantity One program, version 4.6 (Bio-Rad). Each experiment was repeated three times.
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