Yeast protein extraction was performed as described before (Cheong and Klionsky, 2008 (link)). In short, the TCA (Trichloroacetic acid; to a final concentration of 10% for 20 min on ice) precipitated cell pellet was resuspended in 100 μl MURB buffer (50 mM Na2HPO4, 25 mM 2-[N-morpholino]ethanesulfonicacid (MES), pH 7.0, 1% SDS, 3 M urea, 0.5% 2-mercaptoethanol, 1 mM NaN3 and 0.05% bromphenol blue) and disrupted by vortexing with an equal volume of acid-washed glass beads for 5 min. Followed by an incubation at 70°C (1400 rpm) for 10 min. For detection of biotinylation on the acceptor peptide tagged-proteins 10 μg of Streptavidin (2 mg/ml) was added to the MURB buffer resuspended sample. Proteins were separated by NuPAGETM NovexTM 4%–12% gradient or 12% Bis-Tris-protein gels (Invitrogen), transferred onto PVDF membranes (Immobilon®-P) and then analyzed using specific antibodies (see Key Resources Table ) and imaging with an Odyssey® Fc imager (LI-COR) or classical film development. For quantification performed with the Odyssey® Fc imager (LI-COR), proteins were transferred onto low fluorescence PVDF membranes (Immobilon®-FL), and analyzed using specific antibodies followed by fluorescent IRDye® secondary antibodies.
Odyssey fc imager
Manufactured by LI COR
Sourced in United States, Germany, United Kingdom
The Odyssey Fc Imager is a versatile fluorescence imaging system designed for a wide range of applications. It features high-resolution imaging, sensitive detection, and a compact design. The Odyssey Fc Imager is capable of detecting and quantifying fluorescent signals in various sample types, making it a valuable tool for researchers in diverse fields.
Automatically generated - may contain errors
Lab products found in correlation
Image studio software, by LI COR (20 mentions)
Odyssey blocking buffer, by LI COR (14 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (9 mentions)
Protease inhibitor cocktail, by Roche (8 mentions)
Irdye 800cw, by LI COR (7 mentions)
Trans blot turbo transfer system, by Bio-Rad (6 mentions)
Dc protein assay, by Bio-Rad (6 mentions)
Image studio, by LI COR (6 mentions)
Nitrocellulose membrane, by Bio-Rad (6 mentions)
Protease inhibitor, by Merck Group (5 mentions)
Immobilon fl pvdf membrane, by Merck Group (5 mentions)
Ripa buffer, by Thermo Fisher Scientific (5 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Bradford assay, by Bio-Rad (4 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Mini protean tgx gel, by Bio-Rad (4 mentions)
Trans blot turbo system, by Bio-Rad (4 mentions)
Ripa buffer, by Merck Group (4 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (4 mentions)
Ecl kit, by Thermo Fisher Scientific (4 mentions)
209 protocols using odyssey fc imager
1
Yeast Protein Extraction and Detection
2
Western Blot Protein Analysis Protocol
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Total extracts were obtained by scraping cells on plates or resuspending cell pellets in Laemmli buffer (50 mM Tris-HCl pH 6.8, 1.6% Sodium Dodecyl Sulfate, 8% glycerol, 4% β-mercaptoethanol, 0.0025% bromophenol blue) followed by 5 min denaturation at 95 °C.
For western blot analysis, extracts were run on 4–20% Mini-PROTEAN TGX gels (Bio-Rad) in running buffer (200 mM glycine, 25 mM Tris, 0.1% SDS) and transferred onto nitrocellulose membranes (Protran) with a Trans-Blot SD semi-dry transfer cell (Bio-Rad). Total proteins were revealed by Pierce® Reversible Stain (Thermo Scientific). Proteins of interest were probed using the appropriate primary and HRP (Horse Radish Peroxidase)-conjugated secondary antibodies (Supplementary Table2 ), detected using SuperSignal West Pico or Femto chemiluminescence substrates (Pierce) on hyperfilms MP (Amersham) or with Odyssey Fc-imager (LI-COR Biosciences). Alternatively, when fluorescence detection was used instead of chemi-luminescence, total proteins were revealed with REVERT total protein stain, secondary antibodies were conjugated to IRDye 680RD or 800CW (Supplementary Table 2 ) and imaging was performed with Odyssey Fc-imager (LI-COR Biosciences). Levels of proteins of interest were then quantified with Image Studio Lite software using total protein stain for normalisation.
For western blot analysis, extracts were run on 4–20% Mini-PROTEAN TGX gels (Bio-Rad) in running buffer (200 mM glycine, 25 mM Tris, 0.1% SDS) and transferred onto nitrocellulose membranes (Protran) with a Trans-Blot SD semi-dry transfer cell (Bio-Rad). Total proteins were revealed by Pierce® Reversible Stain (Thermo Scientific). Proteins of interest were probed using the appropriate primary and HRP (Horse Radish Peroxidase)-conjugated secondary antibodies (Supplementary Table
3
Rapid Cytoplasmic and Nuclear Protein Extraction
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Cells were rinsed with PBS and lysed using the 0.5% NP-40 (#13021, Millipore Sigma) with HaltTM protease and phosphatase inhibitor cocktail (#78442, Thermo Fisher Scientific). Cytoplasmic and nuclear lysate were prepared using a Rapid, Efficient And Practical (REAP) method [28 (link)]. Briefly, cell pellets were resuspended in ice-cold 0.5% NP-40 in PBS and centrifuged at 4 °C for 10 s (10,000 rpm). The supernatant was removed as cytoplasmic lysate. After the remaining supernatant was removed, the pellet was resuspended in 1 ml of ice-cold 0.5% NP-40 in PBS and centrifuged as above for 10 s and the supernatant was discarded. The pellet was resuspended in 0.5% NP-40 in PBS and designated as nuclear lysate. The BCA method (#23227, Thermo Fisher Scientific) was used for protein quantification. Lysates were boiled for 5 min, resolved using NuPAGE 4–12% SDS–PAGE gels (#NP0335BOX, Thermo Fisher Scientific) and transferred to NC membranes (#IB23002, Thermo Fisher Scientific) using iBlot2TM Blotting System (#IB21001, Thermo Fisher Scientific). Membranes were blocked using InterceptTM Blocking Buffer (#927-60001, LI-COR Biosciences), probed with primary antibodies overnight at 4 °C, and secondary antibodies at room temperature (RT) for 1 h. The immune complexes were visualized using the OdysseyTM Fc Imager (LI-COR Biosciences).
4
Western Blot Analysis of Neuronal Proteins
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Western blots were performed as previously described (Urraca et al., 2018 (link)). Briefly, protein was extracted from 3-week mature neurons using neuronal protein extraction reagent (N-PER) (Fisher Scientific, Waltham, MA) and protease inhibitor cocktail (Roche). Samples were resolved on a NuPage 1.5mm 4–12% Bis-Tris gel (Invitrogen, Carlsbad, CA) according to manufacturer instructions and transferred to an InvitrolonTM -PVDF membrane (Invitrogen, Carlsbad, CA). The membrane was blocked using Odyssey Blocking Buffer (Licor, Lincoln, NE) for 1 h and incubated overnight at 2–8°C in primary antibody with agitation. Primary antibodies used: anti-MAP2 (Santa Cruz, sc-32791), anti-Nestin (Santa Cruz, sc-23927), and anti-GABA A receptor beta 3 (Abcam, ab98968). anti-GAPDH (Abcam, ab157156) was used as a protein loading control. Blots were incubated at room temperature for 1 h in secondary antibodies for both the 700 and 800 channels using Li-Cor IR secondary antibodies (Licor, 926-32212 and 926-68074). Blots were imaged on a Li-Cor OdysseyTM Fc Imager. Both the 700 and 800 channels were exposed for 2 min.
5
Immunoblotting of Cyanobacterial Photosynthetic Proteins
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Cell samples were taken from the Synechocystis culture at different growth phases to a final OD730 of 60 in 1 ml. Samples were spun down (10 min, 3500 g) and resuspended in 50 mM MES buffer, pH 6.5. The cells were broken by four passages through a French pressure cell at 12,000 p.s.i. The protein concentration of the cell-free extracts was determined with a Pierce BCA assay and 10 µg of protein was loaded onto 15% SDS-PAGE gels. Gels were run for 2 h at 10 mA per gel and proteins were transferred to nitrocellulose membranes by wet blotting at 50 mA overnight. Blots were stained with antibodies elicited against AtpB and PsbA or PsaC. Blots with antibodies against RbcL were conducted without AtpB because these proteins are of approximately the same size. For each antibody at least 3 blots were made. GARPO (goat anti-rabbit peroxidase) was used as the secondary antibody. Visualization was achieved using a supersensitive chemo-luminescence kit (Thermo) and a LiCor Odyssey FC Imager. Band intensity was determined in triplicate using ImageJ software. Band intensities of the different proteins were normalized to the band intensity of AtpB. RbcL band intensity was normalized to AtpB from AtpB-PsbA blots which were conducted simultaneously.
6
Purification of Soluble PCDH1 Variants
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Soluble PCDH1 variants cloned into pcDNA3.1 (see above) were expressed in 293 F cells in shaker flasks by transient transfection with linear polyethyleneimine (Polysciences) and purified by nickel-chelation chromatography. Cell cultures were incubated at 37 °C and 8% CO2 for six days post-transfection. Cell supernatants were clarified and stirred overnight at 4 °C with proteinase inhibitor (Sigma-Aldrich) and nickel-NTA resin (Qiagen) at 0.3 mL packed resin per 50 mL cell supernatant. Nickel-NTA beads were then collected, washed with phosphate buffer saline (PBS) containing 50 mM imidazole, and eluted with PBS containing 250 mM imidazole. The eluted protein was buffer-exchanged with PBS, concentrated, and stored in aliquots at −80 °C. The purity of the secreted PCDH1 variants was determined by size-exclusion chromatography (SEC) and/or either SDS-PAGE or Native-PAGE gels, stained with Bio-Safe™ Coomassie G-250 Stain (Bio-Rad) and imaged on a LI-COR OdysseyⓇ Fc Imager (LI-COR Biosciences, LICOR Image Studio software, V.1.0.19). For analytical SEC, a Superdex S200 (10/300) column was equilibrated in PBS and calibrated with Gel Filtration Standard (Bio-Rad) composed of thyroglobulin (MW 670 kDa), bovine γ-globulin (MW 158 kDa), chicken ovalbumin (MW 44 kDa), horse myoglobin (MW 17 kDa), and vitamin B12 (MW 1.35 kDa).
7
Western Blot Analysis of Proteins
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Cell lysates were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% polyacrylamide gels and transferred to nitrocellulose membrane in Tris-Glycine-ethanol buffer (25 mM Tris Base, 192 mM Glycine, 20% Ethanol) for 90 minutes at 400mAmps. Transfer of proteins was confirmed with staining with Ponceau S Red (Sigma). Nonspecific binding sites were blocked with 4% skim milk or BSA in PBS. Blots were incubated overnight at 4 °C in primary antibody diluted in 1% skim milk or BSA in PBST (PBS with 0.1% Tween 20). After overnight incubation blots were washed in PBST and incubated in species specific secondary antibody conjugated to horseradish peroxidase diluted 1:5000 in 1% skim milk in PBST. Bound antibodies were detected with Enhanced Chemiluminiscence (ECL, Perkin Elmer) and exposure on Li-Cor Odyssey Fc Imager. Where appropriate, digital images were analysed using ImageJ to estimate protein levels relative to tubulin and values expressed as arbitrary units normalised to tubulin.
8
Kidney Protein Expression Analysis
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The expression of podocin, nephrin, pAkt and pGSK3β in the kidney cortices was determined by western blot analysis. Equal amount of protein (20 μg for podocin and nephrin, and 100 μg for pAkt and pGSK3β) was loaded at 4–20% SDS-PAGE, transferred to activated PVDF membrane, and immunoblotted with anti-podocin, anti-nephrin, anti-phospho-Akt (Ser-473) and anti-phospho-GSK3β (Ser9) respectively. β-Actin was used as loading control for podocin and nephrin and total Akt and total GSK3β were used to normalize pAkt and pGSK3β. For dot blot analysis, an equal amount of protein (10 μg) was directly spotted onto the activated PVDF membrane. The membrane was then incubated with specific anti-megalin or anti-cubilin antibody in 5% BSA-PBST (phosphate buffered saline containing 0.05% tween-20) overnight at 4 °C. The membrane was washed with PBST (5 mL, 10 min × 3), immunoprobed with relevant secondary HRP-conjugated antibodies, namely goat-anti-mouse IgG secondary antibody, goat-anti-rabbit IgG secondary antibody, for 1 h at room temperature, washed with PBST and the electrochemiluminescence signal was recorded and the bands density was analyzed (BioRad ChemiDoc MP Imaging System or Li-Cor Odyssey Fc Imager). The original blots are provided in SI Fig. S1 -4 .
9
Western Blot Immunodetection of Immune Sensors
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Samples were lysed in a lysis buffer (1% TritonX-100, 10 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, 30 mM Na4P2O7, 50 mM NaF, 0.1 mM Na3VO4) with the addition of protease and phosphatase inhibitors (1%). Protein content was determined and equal amounts of protein were loaded and electrophoresed on a 4–20% precast polyacrylamide gel (Bio-Rad Laboratories AB, Solna, Sweden). The fractionated proteins were blotted on a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories AB, Solna, Sweden) and this was followed by blocking of the membrane in 5% (w/v) milk in Tris-buffered saline Tween-20 and overnight incubation with primary antibodies at 4 °C: anti GAPDH mAb, anti TLR3 mAb, anti RIG-I Rabbit mAb and anti MDA5 Rabbit mAb (Cell Signaling Technology, Leiden, The Netherlands). Membranes were washed and incubated for 1 h with secondary antibodies: anti Rabbit IgG HRP-linked Ab (Cell Signaling Technology, Leiden, The Netherlands). Chemiluminescent detection was performed using Clarity MaxTM Western ECL Substrate (Bio-Rad Laboratories AB, Solna, Sweden) and immunoblots were visualized by LI-COR Odyssey Fc Imager (LI-COR Biosciences, Lincoln, NE, USA) and Image Studio (v3.1.4; LI-COR Biosciences, Lincoln, NE, USA).
10
Quantification of R-Loop Formation
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Genomic DNA was extracted from HeLa cells using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich). Genomic DNA (1 μg) was spotted to Amersham Hybond-N+ positively charged nylon membrane presoaked in PBS using the 96-well Bio-Dot Microfiltration apparatus (Bio-Rad). DNA was UV-crosslinked to the membrane with 120 mJ/cm2 using a UV TL-2000 Translinker. Membranes were blocked for 1 h in PBS with 5% fat-free milk (Sigma-Aldrich) and 0.1% Tween-20 (PBS-T) before incubation with mouse S9.6 (anti-R-loop) antibody (1 μg/ml; 1:1,000 dilution) in blocking buffer overnight at 4°C. Membranes were washed three times for 10 min in PBS-T before incubation with goat anti-mouse HRP secondary antibodies (1:5,000 dilution) in PBS-T for 1 h at room temperature. After 3 × 10 min washes in PBS-T membranes were prepared for chemiluminescent signal detection with SuperSignal West Pico Chemiluminescent substrate kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Signals were detected and densitometric quantification of dots were performed on a LI-COR Odyssey Fc Imager (LI-COR).
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