Total protein and Western blots were performed as already published [8] . 25 µg of total protein was used for SDS-PAGE loading. All blots were done in triplicates and analysed with the INTAS Chemostar.
Chemostar
Manufactured by Intas
Sourced in Germany
ChemoStar is a laboratory equipment designed for chemical analysis and testing. It provides precise and reliable results for various chemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Hrp coupled secondary antibody, by Cell Signaling Technology (2 mentions)
Bl21 codonplus de3 rp, by Agilent Technologies (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Western lightning plus ecl reagent, by PerkinElmer (1 mentions)
C reactive protein (crp), by Proteintech (1 mentions)
Apoa 1, by Proteintech (1 mentions)
Clon 2f2, by Nanotools (1 mentions)
Ab2828, by Abcam (1 mentions)
Benzonase, by Thermo Fisher Scientific (1 mentions)
Anti mecp2, by Abcam (1 mentions)
P044701 2, by Agilent Technologies (1 mentions)
Anti asyn, by BD (1 mentions)
Ecl western blotting detection system, by GE Healthcare (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Chemocam system, by Intas (1 mentions)
8 protocols using chemostar
1
Total Protein and Western Blot Analysis
2
Affinity Purification of HA-Tagged Proteins from Yeast and Bacterial Extracts
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GST- and HA-modified proteins were synthesized in E. coli strain BL21-CodonPlus DE3-RP (Agilent), overproducing selected tRNAs to avoid poor translation.
Crude protein extracts from induced E. coli transformants were prepared by sonication. Similar amounts of released GST fusion proteins (according to GST enzyme assays) were bound to glutathione (GSH) sepharose and incubated with yeast or bacterial total protein extracts containing HA fusions of Sin3, Cyc8 or Tup1. Washing conditions and elution of GST fusions together with prey proteins using free GSH have been described (Wagner et al. 2001 (link)). Eluted proteins were analyzed by SDS/PAGE and subsequently transferred to a PVDF membrane. HA-tagged proteins could be visualized by treatment with anti-HA-peroxidase conjugate (monoclonal antibody 12CA5 conjugate; Sigma-Aldrich) and POD chemiluminescent substrate, using a digital imager (ChemoStar, Intas).
Crude protein extracts from induced E. coli transformants were prepared by sonication. Similar amounts of released GST fusion proteins (according to GST enzyme assays) were bound to glutathione (GSH) sepharose and incubated with yeast or bacterial total protein extracts containing HA fusions of Sin3, Cyc8 or Tup1. Washing conditions and elution of GST fusions together with prey proteins using free GSH have been described (Wagner et al. 2001 (link)). Eluted proteins were analyzed by SDS/PAGE and subsequently transferred to a PVDF membrane. HA-tagged proteins could be visualized by treatment with anti-HA-peroxidase conjugate (monoclonal antibody 12CA5 conjugate; Sigma-Aldrich) and POD chemiluminescent substrate, using a digital imager (ChemoStar, Intas).
3
Quantitative Western Blot Analysis of Complement Proteins
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Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, #10600003). Membranes were blocked in TBST + 5% BSA, incubated in primary antibody in TBST + 5% BSA o/n at 4 °C. After incubation with HRP-coupled secondary antibodies (Cell Signaling Technology, #7074, #7076 S), blots were subjected to ECL reaction. Images were acquired using ChemoStar (INTAS Science Imaging Instruments GmbH) and contrast was adjusted using ImageJ (version 1.52d). The band with the strongest signal was set to maximum (black). Quantification of Western blot signals was performed with ImageJ (version 1.52.d). Primary antibodies and dilutions were: C1qA (#11602-1-AP; 1:2000), C1qB (#16919-1-AP; 1:2000), C1qC (#66268-1-Ig; 1:4000), C1R (#17346-1-AP; 1:2000), C1S (#14554-1-AP; 1:4000), C3/C3b/C3 (#21337-1-AP; 1:1000), CFP (#17192-1-AP; 1:1000), CRP (#66250-1-Ig; 1:10000), F2 (#24295-1-AP; 1:5000), F7 (#23058-1-AP; 1:1000), FGG (#15841-1-AP; 1:2000), MBL2 (#24207-1-AP; 1:2000), SAP (#20773-1-AP; 1:2000) (all from Proteintech).
APOB (#sc-393636; 1:200) and MASP-1/3 (#sc-166815; 1:200) were from Santa Cruz Biotechnology.
APOB (#sc-393636; 1:200) and MASP-1/3 (#sc-166815; 1:200) were from Santa Cruz Biotechnology.
4
Bocaviral Capsid Protein Detection
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Western blot analysis was performed as previously described.94 (link) Briefly, 10 μL from each iodixanol-purified virus stock was mixed with an equal volume of 2× SDS sample loading buffer and boiled for 5 min at 95°C. Then, the samples were separated on 8% SDS-PAGE gels and transferred to a nitrocellulose membrane (NeoLab, Heidelberg, Germany) via semi-dry transfer. Membranes were blocked with 5% milk (Roth, Karlsruhe, Germany) for 1 h at RT and incubated overnight with an anti-VP polyclonal primary rabbit antibody (1:1,000 dilution) recognizing the three bocaviral capsid proteins VP1, VP2, and VP3 (kind gift from Maria Söderlund-Venermo, University of Helsinki). For detection, a horseradish peroxidase-conjugated secondary donkey anti-rabbit antibody (GE Healthcare, Chicago, IL, USA; NA934V) was used at a 1:10,000 dilution. To visualize protein bands, Western Lightning Plus-ECL reagent (PerkinElmer, Waltham, MA, USA) was used, and the emitted signal was detected with a chemiluminescence imager (Intas ChemoStar, Göttingen, Germany).
5
Quantifying ApoB, ApoE, ApoAI, and CRP Levels
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As published before (25 (link)), proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, #10600003). Membranes were blocked in TBST + 5% BSA, incubated in primary antibody in TBST + 5% BSA o/n at 4°C. After incubation with HRP-coupled secondary antibodies (Cell Signaling Technology, #7074, #7076S), blots were subjected to ECL reaction. Images were acquired using ChemoStar (INTAS Science Imaging Instruments GmbH) and contrast was adjusted using ImageJ (version 1.52d). The band with the strongest signal was set to maximum (black). Primary antibodies and dilution were: ApoB (#sc-393636; 1:200) from Santa Cruz Biotechnology, ApoE (Proteintech, #66830-1-Ig, 1:20000), ApoAI (Proteintech, #14427-1-AP, 1#2000), and CRP (Proteintech, #66250-1-Ig; 1:10000).
6
Urine-based Western Blot Analysis
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Western blot analysis was performed from 50 µl urine samples. Urine was centrifuged and washed twice with PBS, the sediment fragments were then incubated with ice‐cold RIPA buffer for 30 min on ice, and proteins were isolated. For the analysis of Mxi‐2, a commercially available antibody from nanoTools (clon 2F2) was used and tested for specificity with the provided positive control lysate (A431). For the Vim3 analysis, the already tested and published Vim3 antibody (Davids BioLab) was used.3 ß‐actin was applied according to the manufacturer's protocol and used for neutralization. All blots were done in triplicates and analyzed with INTAS Chemostar.
7
Lateral Flow Assay for Biomarker Detection
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The lateral flow assay was performed as exemplified in Figure 4 . HF 180 was used as analytic membrane. Antibodies were incubated overnight at room temperature and in the dark. As a conjugation pad, the GDFX membrane was used and incubated with the corresponding antibody for 3 h in a dry environment at 37°C. Finally, the lateral assay was set up and the urine sample (50 µl) was incubated for 10 min. For the analysis, fluorescent secondary antibody was used. All lateral flow assays were done in triplicates and analyzed with INTAS Chemostar.
8
Immunoblotting for α-synuclein and MeCP2
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Protein extracts were prepared by resuspending cells in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% Triton × 100, 10 mM MgCl 2 , 1× Halt protease inhibitor-cocktail (Thermo Fisher), 1 μl/ml Benzonase (Thermo Fisher). After incubation on ice for 1 h, 50 μg of protein per lane was separated by SDS gel electrophoresis, transferred to nitrocellulose, and detection was performed with the ECL Western blotting detection system (GE Healthcare) and secondary antibodies conjugated with horseradish peroxidase. The following antibodies and dilutions were used: anti-a-syn, 1:2000 (610786, BD Bioscience); anti-MeCP2, 1:1000 (ab2828, Abcam); anti beta-actin, 1:10000 (A5441, Sigma); goat anti-mouse, 1:2000 (P044701-2, DAKO); and goat anti-rabbit, 1:3000 (7074, Cell Signaling). Immunoreactive signals were detected using the Intas ChemoCam system (Intas) and the software ChemoStar (Intas). Quantification of the signal intensities was performed using ImageJ.
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