The identity of VP4∗ was analyzed by western blot as follows: the P[8], P[6], P[4]-VP4∗ proteins were separated by SDS-PAGE, then transferred to nitrocellulose membrane. After blocking, the membrane was incubated at 37°C with 1:2000 diluted horseradish peroxidase (HRP)-conjugated anti-VP4 antibody 9C4 (9C4-HRP) for 1 h. The broadly reactive antibody 9C4 was screened in our previous studies using hybridoma technology, and the mouse were immunized with his-tagged VP4∗ proteins. Finally, the Western Bright™ ECL substrate (Advansta, Menlo Park, CA) was add to the membrane following five times washing with Phosphate Buffered Saline-Tween (PBST). The membrane was imaged immediately with ImageQuant LAS4000 (GE Healthcare Life Sciences, Pittsburgh, PA).
Westernbright ecl substrate
Manufactured by Advansta
Sourced in United States
WesternBright ECL substrate is a chemiluminescent substrate used for the detection of proteins in Western blotting applications. It is designed to provide a sensitive and reliable signal for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti goat igg hrp sc 2354, by Santa Cruz Biotechnology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Immobilon p, by Merck Group (2 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Anti p70s6k, by Cell Signaling Technology (1 mentions)
Anti phospho p70s6k, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti phospho p38 mapk, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Anti il 1β, by R&D Systems (1 mentions)
Anti p38 mapk, by Cell Signaling Technology (1 mentions)
Anti hif 1α, by Cell Signaling Technology (1 mentions)
Anti phospho nf κb p65, by Cell Signaling Technology (1 mentions)
Anti hif 1α, by Novus Biologicals (1 mentions)
Immobilon western chemiluminescent substrate, by Merck Group (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti iκbα, by Cell Signaling Technology (1 mentions)
Gel doc, by Bio-Rad (1 mentions)
19 protocols using westernbright ecl substrate
1
VP4* Protein Identity Analysis
2
Western Blot Analysis of Cellular Signaling
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Protein samples from cultured cells were prepared by direct lysis in 5× SDS sample buffer, followed by heating at 95 °C for 5 min. Samples were resolved on 8–12% SDS–polyacrylamide gels followed by transfer to PVDF membranes. Membranes were blocked in 5% (w/v) dried milk in Tris-buffered saline/Tween (TBST) for 1 h at room temperature, after which they were incubated with primary antibody, followed by the correct horseradish-peroxidase-conjugated secondary antibody. Blots were developed using Western Bright ECL substrate (Advansta) or Immobilon Western chemiluminescent substrate (Millipore) using a Bio-Rad Gel Doc. Antibodies used were anti-HIF-1α (catalogue no. 14179), anti-IκB-α (9242), anti-phospho-NF-κB-p65 (3033), anti-NF-κB-p65 (8242), anti-phospho-p38 MAPK (9211), anti-p38 MAPK (catalogue no. 9212), anti-phospho-ERK (9101), anti-ERK (4695), anti-phospho-p70-S6K (catalogue no. 9205), anti-p70-S6k (9202), all purchased from Cell Signaling Technology, anti-IL-1β (R&D Systems, AF401-NA), anti-β-actin (Sigma-Aldrich, AC-74), anti-FLAG (Sigma-Aldrich, F1804), L-2HGDH (Antibody Genie, CAB7996), and D-2HGDH (Antibody Genie, CAB16213). anti-HIF-1α from Novus (catalogue no. NB100-449) was used for human blots. Secondary antibodies used were horseradish-peroxidase-conjugated anti-mouse IgG, anti-goat IgG and anti-rabbit IgG (Jackson ImmunoResearch).
3
Immunoblotting Analysis of Peroxisomal Proteins
4
Extraction and Analysis of D. indusiata Mushroom
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The fruiting body of the mushroom D. indusiata was obtained from Anhui Joy Lok Food Co., Ltd., Ningde, Fujian Province, China. The DNA extraction kit (QIAamp DNA Stool Mini Kit) and gel purification kit (Agencourt AMPure XP 60 mL Kit) were from Qiagen (Hilden, Germany) and Beckman Coulter (Brea, CA, USA), respectively. The primary antibodies β-actin, claudin-1, occludin, zonula occludens-1 (ZO-1), secondary antibodies, and the Radioimmunoprecipitation assay (RIPA) buffer were from Proteintech (Wuhan, China). The horseradish peroxidase-conjugated secondary antibody was from ZSGB-BIO (Beijing, China). The bicinchoninic acid (BCA) protein assay kit was from Pierce Rockford, IL, USA. Polyvinylidene difluoride (PVDF) membranes were from Immobilon TM-P, Millipore, Massachusetts, USA, and WesternBright™ ECL substrate was from Advansta, Inc., Menlo Park, CA, USA. The ELISA kits were purchased from Shang Hai Lengton Bioscience Co, Ltd., Shanghai, China. All the other chemicals used in this study were of analytical grade and purchased from standard commercial sources.
5
Western Blot Analysis of EV Biomarkers
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Ten micrograms of total protein of EV suspension were loaded on a 4–12% SDS-PAGE Gel (Invitrogen, Waltham, MA, USA, #NP0322) under reducing (Alix, ApoB100) or non-reducing (CD9, CD63, ApoA1) conditions in the presence of 100 mM dithiothreitol. Primary antibodies against Alix (Cell Signaling, Leiden, Netherlands, #2171), CD9 (System Bioscience, Palo Alto, USA, #EXOAB-CD9A-1), and CD63 (BioLegend, San Diego, CA, USA, #353005, clone H5C9) were used to screen for EV biomarkers, while ApoA1 (Santa Cruz, Heidelberg, Germany, #sc-376818) and ApoB100/48 (Santa Cruz, #sc-393636) were detected to monitor the depletion or co-enrichment of lipoproteins. Each antibody was used diluted 1:1000 in 1% BSA in PBST (PBS + 0.1% Tween 20). HRP-conjugated anti-rabbit (Biorad, Hercules, CA, USA, #170-5046) and anti-mouse (Biorad, #170-5047) were used as secondary antibodies. Detection was performed via enhanced chemiluminescence (ECL) using WesternBright ECL substrate (Advansta, San Jose, USA, #K-12045-D20) on a ChemiDoc device (BioRad). Digital images were automatically white-corrected via the GIMP v2.8.
6
Western Blot Analysis of Protein Extracts
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Worm samples were prepared as follows: The cellular fraction (50 μg) was mixed with 1× Laemmli. For yeast protein extracts, the same amount of protein (based on their OD600 at the time of collection) was loaded into each well.
Samples were separated by electrophoresis in 1× SDS-tris-glycine buffer on precast 4 to 20% TGX gels (Bio-Rad). Proteins were transferred onto polyvinylidene difluoride membrane (activated in methanol for 20 s) for 1 hour at 4°C at 100 V in 1× tris-glycine buffer containing 20% methanol. Membranes were blocked for 1 hour in 1× tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% milk; primary antibodies were added to the same buffer and incubated overnight at 4°C. Membranes were then washed in 1× TBST and incubated with the secondary antibody in TBST containing 5% milk for 1 hour at room temperature. After washing, membranes were incubated with WesternBright ECL substrate (Advansta) and developed using a ChemiDoc system (Bio-Rad). Primary and secondary antibodies are listed in table S10.
Samples were separated by electrophoresis in 1× SDS-tris-glycine buffer on precast 4 to 20% TGX gels (Bio-Rad). Proteins were transferred onto polyvinylidene difluoride membrane (activated in methanol for 20 s) for 1 hour at 4°C at 100 V in 1× tris-glycine buffer containing 20% methanol. Membranes were blocked for 1 hour in 1× tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% milk; primary antibodies were added to the same buffer and incubated overnight at 4°C. Membranes were then washed in 1× TBST and incubated with the secondary antibody in TBST containing 5% milk for 1 hour at room temperature. After washing, membranes were incubated with WesternBright ECL substrate (Advansta) and developed using a ChemiDoc system (Bio-Rad). Primary and secondary antibodies are listed in table S10.
7
Western Blot Analysis of GILT Expression
8
Quantifying Protein Expression in Immune Cells
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Hippocampal lysate was assessed for expression of high-mobility group protein B1 (HMGB1); BMDM lysate was evaluated for expression of TLR4 and IFNγ receptor (IFNγR1). Briefly, samples were equalized for protein concentration, boiled in gel-loading buffer and separated by gel electrophoresis on 10% sodium dodecyl sulphate-polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and incubated with anti-HMGB-1 antibody (anti-rabbit 1:2,000; Abcam, Cambridge, UK), TLR4 (anti-rabbit 1:1,000; Abcam, Cambridge, UK), IFNγR (anti-rabbit 1:1,000; Abcam, Cambridge, UK) or anti-β-actin antibody (mouse monoclonal; 1:5,000; Sigma-Aldrich, Gillingham, Dorset, UK). Membranes were incubated with horseradish peroxide-conjugated secondary antibodies (1:5,000; Jackson Immunoresearch, West Grove, PA, USA), and bands were visualised using WesternBright ECL Substrate (Advansta, Menlo Park, CA, USA). Images were captured using a Fujifilm LAS-4000 (Brennan and Co, Dublin, Ireland). Densitometry was performed using ImageJ software (http://rsb.info.nih.gov/ij/ ).
9
Western Blot Immunodetection Reagents
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The chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) except those specifically indicated below. Anti-3-nitrotyrosine antibody (9691S), H2A.X Rabbit (2595S) and Phospho-Histone H2A.X Rabbit (9718S) were purchase from Cell Signalling (Danvers, MA, USA). The anti-4-hydroxynonenal (AB5605) antibody was purchased from Millipore Corporation (Billerica, MA, USA). Goat anti-rabbit IgG-HRP (A2315) and mouse anti-goat IgG-HRP (Sc-2354) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The Western Bright ECL substrate was purchased from Advansta (Menlo Park, CA, USA).
10
Quantification of DNA Damage via Slot Blot
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Equal amounts of isolated DNA were diluted in TE10/1-buffer (pH 8), incubated for 5 min in boiling water and cooled down for 2 min on ice. Samples were spotted on positively charged nitrocellulose membrane (GE Healthcare, Little Chalfont, UK) using a slot–lot chamber coupled to a vacuum manifold. The membrane-bound DNA was denatured for 45 min on Whatmann paper soaked with 0.4 N NaOH. Membranes were blocked overnight in 5% skim milk in TBS-Tween-20 (0.5%; TBS-T) at 4 °C. Membranes were incubated for 2 h at 4 °C with a HRP-conjugated thymine dimer antibody (Kamiya Biomedical Company, Tukwila, WA, USA) in 5% skim milk/TBS-T. Membranes were washed and signals were detected using the WesternBright ECL substrate (Advansta, Menlo Park, CA, USA). The signal intensity of the irradiated control sample was defined as 100%. A serial dilution of this sample was spotted and stained for CPDs to ensure linearity of the signal intensity (example shown in Supplementary Figure S5 ). The SWB-based CPD detection in sham-exposed KC did not produce any detectable signals and therefore is not shown.
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