U937 cells were lysed, and proteins were separated on 10% SDS-polyacrylamide gels and transferred onto Immobilon polyvinylidene difluoride membranes (Millipore, Billerica, MA) together with dual color precision plus protein standards (Bio-Rad, Hercules, CA). Membranes were blocked with 5% BSA diluted in PBS and incubated with polyclonal rabbit antibodies to iPLA2 (1 : 5000; Sigma-Aldrich) or mouse monoclonal antibodies to β-actin (1 : 50000; Sigma-Aldrich). Bound primary antibodies were detected with secondary horse radish peroxidase-labeled antibodies (1 : 5000; Dako, Glostrup, Denmark), lumi-light substrate (Roche, Mannheim, Germany), and high-performance chemiluminescence films (GE Healthcare Bio-Sciences, Uppsala, Sweden).
High performance chemiluminescence film
Manufactured by GE Healthcare
Sourced in United States, Germany, United Kingdom
High-performance chemiluminescence film is a laboratory equipment used for the detection and imaging of chemiluminescent signals. It is designed to capture and record low-level light emissions generated in various biochemical and molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus western blotting detection system, by GE Healthcare (4 mentions)
Immobilon polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Precision plus protein dual color standard, by Bio-Rad (3 mentions)
Lumi light substrate, by Roche (3 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Nitrocellulose membrane, by Merck Group (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Amersham ecl western blotting analysis system, by GE Healthcare (2 mentions)
Amersham ecl western blotting, by Cytiva (2 mentions)
Mini protean 2 cell and system, by Bio-Rad (2 mentions)
Beta actin ac 74, by Merck Group (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Bromophenol blue, by Santa Cruz Biotechnology (2 mentions)
Enhanced chemiluminescence system, by GE Healthcare (2 mentions)
Amersham ecl, by Cytiva (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Carl Roth (2 mentions)
Tris glycine sds buffer, by Thermo Fisher Scientific (1 mentions)
Novex wedgewell 4 20 tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Occludin, by Thermo Fisher Scientific (1 mentions)
Precision protein streptactin hrp conjugate, by Bio-Rad (1 mentions)
34 protocols using high performance chemiluminescence film
1
Western Blot Analysis of iPLA2 Protein
2
Protein Expression Analysis by Western Blot
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Total protein extracts, respectively, biotinylated protein extracts were loaded in equal amounts per lane on Novex™ WedgeWell™ 4–20% Tris‐Glycine Gel (Invitrogen, Carlsbad, USA) and after electrophoresis using Tris‐Glycine SDS buffer (invitrogen, Carlsbad, USA) transferred to PVDF membranes (Millipore, Bedford, USA). The membranes were blocked for 1 hr at room temperature in 3% skimmed milk (BIO‐RAD, Hercules, USA). The primary antibodies Zo‐1 (rabbit), (1:200, Invitrogen, Carlsbad, USA), occludin 1:200 (rabbit), (Invitrogen, Carlsbad, USA) and phospho‐caveolin‐1 (rabbit) (1:2000, abcam, Cambridge, UK) were applied to the membranes and incubated at 4°C overnight. Beta‐actin antibody was applied as loading control. After three times wash with TBS/T buffer, the secondary antibodies goat anti‐rabbit lgG‐HRP (Santa Cruz biotechnology, Santa Cruz, USA) (1:5000 diluted) and Precision ProteinTM StrepTactin‐HRP Conjugate (BIO‐RAD, Hercules, USA) (1:10000 diluted) were applied to the membranes and incubated for 1 h at room temperature. The membranes were exposed to Pierce ECL Western Blotting Substrate (Thermo, Rockford, USA) and signals detected by High performance chemi‐luminescence films (GE healthcare, little Chalfont, UK) for further analysis using Image Studio Lite (LI‐COR, Lincoln, US).
3
Western Blot and Immunoprecipitation Protocols
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Cells were lysed with RIPA-P buffer. Proteins were separated using 10 % SDS-polyacrylamide gels. After semi-dry blotting (Bio-Rad) onto PVDF membranes (GE Healthcare) and blocking, the blots were incubated with primary antibodies overnight at 4°C. Subsequently, blots were incubated with secondary antibodies, and for signal detection, SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) was used. Signal detection was performed with high-performance chemiluminescence films (GE Healthcare) and Curix 60 autoprocessor (Agfam). For IP experiments, U937 Lyve-1 cells were cultured in FCS-free medium overnight. Lysates were prepared using DISC buffer. 1 μg anti-Lyve-1 biotin antibody (R&D systems) and 40 μl Protein G-agarose (Sigma-Aldrich) were used per 4 mg protein. The mixture was incubated overnight at 4°C. After washing, the samples were boiled in 1x Laemmli buffer and separated on SDS-polyacrylamide gels and subjected to analysis by Western blot or Coomassie staining. Antibodies are listed in Supplementary Table 2 .
4
Quantification of uPARAP and MR in Macrophages
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Protein concentrations in cell lysates from bone marrow-derived macrophages were first determined using a BCA protein concentration kit (Bio-Rad). For the analysis of uPARAP and MR expression, 12 μg of protein from each lysate was separated by SDS-PAGE and blotted onto a PVDF membrane. 2% BSA solution was used for blocking. Primary mouse anti-uPARAP mAb 2h9 (0.5 μg/mL) [61 (link)] or rat anti-MR (1 μg/mL, clone MR5D3, Bio-Rad) diluted in a 0.1% Tween20 PBS solution were applied overnight at 4 °C. Secondary Rabbit-anti-mouse or Rabbit-anti-Rat HRP conjugated antibodies (Dako) were applied at a 1:3000 dilution in 0.1% Tween20 PBS solution. ECL western blotting detection reagents and High Performance Chemiluminescence films (GE Healthcare) were used for development. For loading controls, 10 μg of protein from each lysate was separated by SDS-PAGE and stained with Coomassie brilliant blue to visualize proteins in each lysate.
5
Western Blotting of P2Y Receptors
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Protein extracts of U937 cells were separated on 12% SDS-polyacrylamide gels under reducing conditions along with dual color precision plus protein standards (Bio-Rad, Hercules, CA, USA) and transferred onto Immobilon polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% BSA diluted in PBS and incubated with polyclonal rabbit antibodies to P2Y1, P2Y11 (LS-C163318; 1:1000 and LS-C200442; 1:500; LifeSpan Biosciences via Biozol, Eching, Germany) or mouse monoclonal antibodies to β-actin (1:50000; A2228, Sigma-Aldrich). Primary antibodies were detected with horse radish peroxidase-labeled goat anti-rabbit Ig and rabbit anti-mouse Ig secondary antibodies (1:5000; Dako, Glostrup, Denmark), Lumi-Light substrate (Roche, Mannheim, Germany) and High Performance Chemiluminescence Films (GE Healthcare Bio-Sciences, Uppsala, Sweden). Documentation and densitometry were performed using a digital gel documentation system (Biozym, Hessisch Oldendorf, Germany). Western blot analysis of IL-1β in concentrated cell culture supernatants was performed as described before [8 (link)].
6
Quantification of Protein Expression in U937 Cells
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U937 cells were lysed and the protein concentration was assessed using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). 10 µg of protein per sample was fractionated by SDS-PAGE on 10% gels and transferred to Immobilon polyvinylidene difluoride membranes (Millipore, Billerica). Dual color precision plus protein standards (Bio-Rad, Hercules, CA, USA) were used as molecular weight markers. Membranes were blocked with Roti®-Block (Roth, Karlsruhe, Germany) (CD36), 5% low-fat milk powder (Roth) (β-actin) or 5% BSA (AAT) in PBS and incubated with primary antibodies (anti-AAT 1:20,000, anti-CD36 1:1,000, β-actin 1:50,000) in blocking solution. PBS supplemented with 0.01% Tween-20 was used for washing steps and appropriate secondary antibodies (1:5,000 each) were diluted in PBS, 0.01% Tween-20, 2.5% low-fat milk powder. SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) was used for the detection of CD36, blots were developed using Lumi-Light substrate (Roche, Mannheim, Germany) to visualize β-actin and documented with High Performance Chemiluminescence Films (GE Healthcare).
7
Western Blot Analysis of DENV Antigens
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Laemmli sample buffer was added to protein samples and after boiling for 5 minutes the samples were loaded and separated by SDS-PAGE on 12% polyacrylamide gels following the protocol described by Laemmli [38] and stained with Coomassie Blue [39] . For Western blot analysis, gels were transferred onto a nitrocellulose membrane and DomIIIHFBI was detected using a rabbit polyclonal anti-DENV 1+2+3+4 antibody (Abcam) in a dilution 1/833 as the primary antibody and Peroxidase-conjugated AffiniPure Mouse anti-rabbit IgG (Jackson ImmunoResearch) in a dilution 1/4000 as the secondary antibody [40] . Development was carried out with an enhanced chemiluminescent substrate (Thermo Fisher Scientific) and high performance chemiluminescence films (GE Healthcare). For image processing, gels were scanned and then analyzed with the ImageJ (strain Hawaii), DENV-2 (strain NGC), DENV-3 (strain H87), DENV-4 (strain H241) and Yellow fever virus (YFV, vaccine strain 17D-YEL). A selection of paired sera from patients showing monotypic neutralizing antibody pattern was employed [41] . Cut off was determined as the mean + 3 SD of negative samples.
8
Western Blot Analysis of Protein Expression
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For the analysis of protein expression, cell pellets were washed and resuspended in PBS. Cells were centrifuged and pellets were stored at −20 °C. Cell pellets were then lysed in Winman’s buffer containing 1% NP-40, 0.1 M Tris–HCl pH 8.0, 0.15 M NaCl, and 5 mM EDTA, complemented with protease inhibitor cocktail (Roche). The total protein content was quantified using the DC™ Protein Assay kit (Bio-Rad, Hercules, CA, USA) according to manufacturer’s instructions. Protein lysate (20 µg) were loaded on 12% SDS-PAGE gel and transferred into a nitrocellulose membrane (GE Healthcare, Cleveland, OH, USA). The following primary antibodies were used: rabbit anti-PARP-1 (1:2000, sc-7150, Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-Caspase 3 (1:2000, 05-654, Merck Millipore, Darmstadt, Germany), and goat anti-actin (1:2000, sc-1616, Santa Cruz Biotechnology). The corresponding secondary antibodies were: anti-rabbit IgG-HRP, anti-mouse IgG-HRP, or anti-goat IgG-HRP (1:2000, Santa Cruz Biotechnology). The Amersham™ ECL Western Blotting Detection Reagents (GE Healthcare), the High Performance Chemiluminescence Film (GE Healthcare), and the Kodak GBX developer and fixer (Sigma) were used for signal detection [35 (link),36 (link)].
9
Western Blot Analysis of MBP and Actin
10
Immunoblot Analysis of Adhesins
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Bacterial samples from at least five independent experiments, were collected from plate, washed twice in Sia buffer. SDS Page analysis was made using NuPage Novex Bis-Tris protein gels (4–12% or 12%) using 1x MOPS SDS as running buffer (Life Technologies). The gels were either stained with PageBlue Protein stain (Thermo Scientific) or transferred to PVDF membrane using Trans-Blot SD Semi-Dry Transfer Cell (Bio Rad). Immunoblot analysis was performed as previously described44 (link). Antibodies against AlpB (AK262)45 (link), SabA (AK278) and BabA (AK277), were used in combination with secondary anti-rabbit IgG-HRP (P0160, DAKO A/S, Denmark). Blots were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and detected on High Performance Chemiluminescence film (GE Healthcare). BabA, SabA and AlpB protein densities were measured by ImageJ software (a public domain image processing program from the National Institutes of Health, Maryland, USA).The adhesin/AlpB density ratio for each sample was used to calculate the fold difference between the wt and the arsS deletion mutant.
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