The protein was extracted using 1X RIPA extraction buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific) and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The protein concentration was measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). The cell protein was loaded into NuPAGE™ 4–12% Bis-Tris Protein Gels (Invitrogen) and resolved in the PVDF membrane (BioRad) using the Trans-Blot® Turbo™ transfer system (BioRad). The membranes were blocked with 5% nonfat dry milk or 5% BSA in TBST for 1 hour at RT. Membranes were then incubated overnight at 4°C with primary antibodies. After washing with TBST three times, the membranes were incubated with donkey anti-mouse antibody or swine anti-rabbit antibody conjugated to horseradish peroxidase secondary for 1 hour at RT. The Super Signal West Pico Chemiluminescent Substrate Kit (Thermo Fisher Scientific) was used, and the membranes were visualized using ChemiDoc imaging systems (BioRad). Further details of the used antibodies are provided in Supplementary Table 1.
Supersignal west pico chemiluminescent substrate kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, Belgium, Sweden, Panama, China
The SuperSignal West Pico Chemiluminescent Substrate kit is a laboratory reagent used for the detection of proteins in Western blot analysis. It provides a luminescent signal that can be detected by a compatible imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (12 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Trans blot turbo transfer system, by Bio-Rad (5 mentions)
Ripa buffer, by Thermo Fisher Scientific (5 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Las 4000, by Cytiva (5 mentions)
β actin, by Cell Signaling Technology (5 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (3 mentions)
β actin, by Merck Group (3 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (3 mentions)
Bca assay, by Thermo Fisher Scientific (3 mentions)
Horseradish peroxidase coupled secondary antibody, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Thermo Fisher Scientific (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
189 protocols using supersignal west pico chemiluminescent substrate kit
1
Western Blot Protein Detection Protocol
2
Chondrocyte Protein Expression Analysis
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Chondrocyte pellets were dissolved in citrate-EDTA buffer (55 mM/5 mM; pH 6.8) for 10 min at 37 °C and centrifuged at 10 K rpm for 15 s. Then, the cell pellet was washed five times with PBS and resuspended in 200 μL of SDS-NuPAGE buffer for NuPAGE gel (Invitrogen, Grand Island, NY, USA) running. Immunoblotting was performed using monoclonal antibodies against Type Ⅰ collagen, Type Ⅱ collagen (1:1000 dilution; EMD Millipore, Billerica, MA, USA), MMP-13 (Abcam, Cambridge, MA, USA) TGF-β (1:1000 mg/mL; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and PPAR-γ (Abcam, Cambridge, MA, USA). The loading control, β-actin, will be detected using anti-β-actin antibody (1:10,000 dilution; Abcam, Cambridge, MA, USA). Secondary antibodies tagged horseradish peroxidase (HRP) (rabbit for TGF-β 1:20,000 dilution and mouse for type Ⅱ collagen and β-actin, 1:5000 dilution) was used. Proteins separated in the gel and the gel were transferred to nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA) using TE77XP semidry blotter with 10 V for 3 h (Hoefer, Inc., Holliston, MA, USA). Protein band signals on blots were detected on Amersham Hyperfilm which was enhanced chemiluminescence (GE Healthcare Life Sciences, Pittsburgh, PA, USA) using SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific, Rockford, IL, USA).
3
Western Blot Analysis of MDV Infection
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Western blot analyses were performed essentially as previously described (44 (link)). To detect the relative level of MDV infection, mouse mAb H19 (45 (link)) was used at 1:10,000 dilution to detect MDV pp38. To detect RLORF4 tagged with mRFP, anti-mRFP polyclonal antibody (ab62341; Abcam, Cambridge, MA, USA) was used at 1:2000 dilution. Anti-Flag M2 mAb (F1804, Sigma-Aldrich) were used at the manufacturer's recommended dilutions to detect 3 × Flag-tagged pICP27 (UL54). For protein loading controls, anti-GAPDH (GA1R; Thermo Scientific) and anti-β-Actin (ACTNO5; Abcam) mAb were used at their recommended dilutions. Secondary anti-mouse or rabbit IgG-peroxidase conjugate was purchased from GE Healthcare (Piscataway, NJ, USA). The SuperSignal West Pico Chemiluminescent Substrate kit from Thermo Fischer Scientific (Rockford, IL, USA) was used to detect antigens utilizing the manufacturer's instructions. Images were obtained using a FluorChem R imaging system (ProteinSimple, CA, USA) in 8-bit format. Protein bands were quantified using ImageJ software (version 1.6) for densitometric analysis by comparing the relative ratios of viral protein to GAPDH using the technique described on the ImageJ website (https://imagej.nih.gov/ij/docs/ ).
4
Evaluating Immune Signaling Proteins
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The levels of cGAS, STING, HBx, and IRF3 in cells were evaluated by western blotting. Briefly, samples containing an equal amount of protein were separated by SDS-PAGE and blotted onto PVDF membranes. The membranes were blocked with 5% bovine serum albumin and incubated with anti-cGAS, anti-STING, anti-HBx, anti-IRF3, anti-Flag, anti-HA, anti-K48-linked ubiquitin, or anti-β-actin antibodies, followed by incubation with secondary antibodies conjugated with horseradish peroxidase. The proteins of interest were detected using the SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific, USA). The results were recorded by the Bio-Rad Electrophoresis Documentation (Gel Doc 1000, Bio-Rad, USA) and Quantity One Version 4.5.0.
5
Immunoblotting Analysis of Teleost T-cell Markers
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Different amounts of recombinant CD3ε, CD4-1, and CD4-2 protein samples and 1 × 107 cells/mL of cell suspensions from the spleen, head-kidney and peripheral blood resuspended in RIPA buffer were separated on 15% SDS-PAGE under reducing conditions (80 V for 20 min, 120 V for 90 min) [1 (link),12 (link)]. Protein bands in gels were transferred to methanol-activated polyvinylidene fluoride (PVDF) membranes at 50 mA for 90 min. The membranes were blocked with 5% (w/v) skim milk in 1x PBS containing 0.1% (v/v) Tween 20, and then incubated with specific monoclonal antibodies (CD3ε, 4B2; CD4-1, 10F8; CD4-2, 3C8) followed by HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Massachusetts, USA) [1 (link),12 (link)]. These membranes were then stained with a SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific, Massachusetts, USA).
6
SDS-PAGE and Immunoblotting of Biotinylated Proteins
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Purified surface protein fraction for biotinylation (30 µg/lane) was separated on a 4–15% SDS-polyacrylamide gel and transferred onto PVDF (polyvinylidene difluoride) membranes. The membranes were then blocked with 5% non-fat dry milk made in tris-buffered saline containing 0.1% Tween 20 for 2 hrs. Membranes were then incubated with appropriate primary antibodies overnight at 4°C in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBS-T) and 5% nonfat dry milk. Membranes were then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1∶10,000 dilution; Milipore) for 1–2 hrs at room temperature. Proteins were then visualized using SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific). A slo1β4 antibody (1∶200 dilution; Alamone) was used to recognize the N-terminus of β4 subunit, and a mouse monoclonal anti-FLAG M2 antibody (1∶200 dilution; Sigma Aldrich) was used to detect the C-half chimera.
7
Western Blot Analysis of Amyloid-β
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Five microliters of either Aβ1–42 oligomers or Aβ42–1 peptide (80 μM) were mixed with 1 × loading buffer and separated by 15% SDS-PAGE without boiling. Then they were transferred to nitrocellulose membranes. The membranes were blocked for 1 h in a solution of 3% (w/v) BSA diluted in TBS-T buffer (20 mM Tris/HCl [pH 7.4], 150 mM NaCl and 0.1% Tween-20), and incubated overnight with rabbit monoclonal antibody for Aβ1–42 (D9A3A) (Cell Signaling Tecnnology; 1:1000), which was produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human β-amyloid (1–42) peptide. HRP-conjugated goat anti-rabbit IgG (SunShine Bio, Nanjing, Jiangsu, China) was used as the secondary antibody (1:500). Finally, the signals were developed using the Super Signal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific, Waltham, MA). Molecular mass was assessed by the Rainbow molecular weight markers (Bio-Rad, Hercules, CA).
8
Western Blot Analysis of Tissue Proteins
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Tissues were lysed with Thermo Scientific Pierce T-PER Tissue Protein Extraction Reagent (Thermo Scientific., St. Louis, MO, USA) containing cOmplete ULTRA Protease Inhibitor and PhosSTOP Phosphatase Inhibitor Cocktails (Roche, Branchburg, NJ, USA). Proteins were separated by 4 ~ 12% SDS-PAGE gel electrophoresis and transferred to an Immun-Blot PVDF Membrane. The membrane was then incubated with specific primary antibodies, i.e., rabbit anti-TTR antibody (1:1000 dilution; ABBIOTEC., San Diego, CA, USA) or rabbit anti-NPY antibody (1:1000 dilution; ImmunoStar., Hudson, USA), followed by incubation with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (Sigma-Aldrich), and detected by the SuperSignal West Pico Chemiluminescent Substrate Kit (Thermo Scientific). Anti-β-actin antibody was used for loading controls.
9
Quantifying Extracellular Matrix Proteins
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Protein expression levels of COL1A1, MMP-1, MMP-14, and LOX were quantified by immunoblotting. Approximately 100 μg of the whole cell protein lysates from tumor tissue, along with a prestained, broad range standard molecular weight marker were resolved on a 7% polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane overnight after which the membrane was removed, blocked with 5% nonfat milk for 2 h and further incubated overnight with antibody. Antibodies used were rabbit-polyclonal anti-COL1A1 antibody (1:1000; OriGene, Rockville, MD, USA), rabbit polyclonal anti-MMP-1 antibody (1:1000 dilution; Neo BioLab, Woburn, MA, USA), rabbit polyclonal anti-MMP-14 antibody (1:1000 dilution; Neo BioLab, Woburn, MA, USA), mouse monoclonal anti-LOX antibody (1:1000 dilution; GeneTex, Inc., Irvine, CA, USA), or rabbit polyclonal anti-LOX-L1 antibody (1:1000 dilution; Sigma-Aldrich Corp. St. Louis, MO, USA). Horseradish peroxidase-conjugated secondary antibodies were used at 1:2000 dilution. Blots were visualized using the SuperSignal West Pico Chemiluminescent substrate kit (Thermo Scientific, Rockford, IL, USA). The reference band from the molecular weight marker was used to determine the location of the protein of interest.
10
Characterization of Hepatic Microsomal Enzyme Activity
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The hepatic microsomal fractions were characterised for Cyp1a1 enzyme activity using Sudan I oxidation (Stiborova et al. 2002 (link)) and for Cyp1a1 and Cyp1a2 enzyme activities, we used 7-ethoxyresorufin O-deethylation (EROD) (Stiborova et al. 2005 (link)). POR enzyme activity was determined using cytochrome c (Arlt et al. 2003 (link)). Western blotting analysis using 4–12% bis–tris gradient gels and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were carried out as described previously (Kucab et al. 2012 (link)). After migration, the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes and the following primary antibodies were used: anti-Cyp1a1 1:1000 (sc-20772 (H-70), Santa Cruz Biotech), anti-POR (ab39995, Abcam), anti-Cyb5 1:750 (ab69801, Abcam), and anti-Cyb5R 1:1000 (ABIN453978, antibodies-online.com). The antibody to detect glyceraldehyde phosphate dehydrogenase (Gapdh) 1:25,000 (MAB374, Chemicon) was used as loading control. The secondary horseradish peroxidase-linked antibodies were as follows: anti-goat 1:10,000 (sc-2020, Santa Cruz) anti-rabbit 1:10,000 (#170–5046, BioRad). The antigen–antibody complex was visualised using SuperSignal® West Pico Chemiluminescent Substrate Kit (Thermo Scientific).
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