10μg (total extracts) and 20μg (nuclear extracts) of lysates were loaded onto a 4%–12% Bis-Tris SDS-PAGE gel (Life Technologies) and transferred to a PVDF membrane (Bio-Rad). Western blots were performed according to standard procedure. We used the following antibodies: α-AKT (CST, 4685), α-AKT (pS473) (Santa Cruz, sc-7985), α-GR (Santa Cruz, sc-393232), α-CRY1 (Abcam, ab104736), and α-CRY2 (kindly supplied by Katja Lamia). Secondary antibodies were: α-rabbit IgG (Santa Cruz, sc-2317), α-mouse IgG (BioRad, 170–6516) and α-guinea pig IgG (Santa Cruz, sc2438).
Bis tris sds page gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
4–12% Bis-Tris SDS-PAGE gels are precast polyacrylamide gels used for the separation and analysis of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels have a Bis-Tris buffer system and a gradient of 4% to 12% acrylamide concentration, which allows for the effective separation of a wide range of protein sizes.
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Lab products found in correlation
Mst14, by R&D Systems (8 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (6 mentions)
Immobilon p membrane, by Merck Group (6 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Odyssey clx infrared imaging system, by LI COR (4 mentions)
Blotting grade blocker, by Bio-Rad (3 mentions)
Odyssey imaging system, by LI COR (3 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Ecl western blotting substrate, by PerkinElmer (3 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (2 mentions)
Irdye 800cw conjugated goat anti human secondary antibody, by LI COR (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Las 3000, by Fujifilm (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Ecl detection system, by GE Healthcare (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
89 protocols using bis tris sds page gel
1
Protein Expression Analysis in Lysates
2
NFκB Phosphorylation Analysis by Western Blotting
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Protein concentration was determined by the BCA assay and 10-μg of protein was resolved on a 4–12 % Bis-Tris SDS-PAGE gel (Life Technologies) and transferred to PVDF membrane (Bio-Rad). Immunoblotting was performed using anti-phosphorylated NFκB p65 subunit (Cell Signaling Technology, Danvers, MA) and anti-vinculin (Sigma) antibodies diluted in 5 % Blocking Reagent (Bio-Rad) in Tris-buffered saline and 0.1% Tween 20 (TBS-T). After washing with TBS-T, membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Amersham Biosciences, Piscataway, NJ). The membranes were then incubated with ECL Western Blotting Detection reagent (Pierce, Rockford, IL) and processed on Kodak BioMax XAR X-ray film (Fisher). Densitometry was performed using ImageJ.
3
Western and Lectin Blot Protocols
4
SARS-CoV-2 Spike Protein Detection
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24 well plates were seeded with MRC-5 cells (1.25×105 cells/well), and after overnight growth they were transduced with Ad26 vectors encoding SARS-CoV-2 Spike transgenes. Cell lysates were harvested 48 h post transduction and, after heating for 5 min at 85°C, samples were loaded under non-reduced conditions on a precast 4–12% Bis-Tris SDS-PAGE gel (Invitrogen). Proteins were transferred to a nitrocellulose membrane using an iBlot2 dry blotting system (Invitrogen), and membrane blocking was performed overnight at 4°C in Tris-buffered saline (TBS) containing 0.2% Tween 20 (V/V) (TBST) and 5% (W/V) Blotting-Grade Blocker (Bio-Rad). Following overnight blocking, the membrane was incubated for 1 hour with 2.8 μg/ml CR3046. in TBST-5% Blocker. CR3046 is a human monoclonal antibody directed against SARS-CoV Spike and binds to the Spike S2 domain and also cross-reacts with SARS-CoV-2 Spike S2 (unpublished data). After incubation, the membrane was washed three times with TBST for 5 minutes and subsequently incubated for 1 hour with 1:10,000 IRDye 800CW-conjugated goat-anti-human secondary antibody (Li-COR) in TBST-5% Blocker. Finally, the PVDF membrane was washed three times with TBST for 5 minutes, and after drying developed using an ODYSSEY® CLx Infrared Imaging System (Li-COR).
5
SARS-CoV-2 Spike Protein Detection
6
Immunoprecipitation and Western Blot Analysis
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HEK293 cells were cultured in 75 cm2 culture flasks and transfected with 10 µg of the appropriate CFP- and HA-tagged constructs in a 1∶1 ratio using the Lipofectamine reagent according to the manufacturer's instructions (Invitrogen). Cells were solubilized 48 hours post-transfection in a 1 x PBS buffer supplemented with 5 mM EDTA, 1% Triton X-100 and a complete protease inhibitor mixture (Roche Diagnostics). Precipitation of the protein complexes from the soluble cell fraction was performed with GFP antibodies (Abcam, Cambridge, UK) and Protein G Agarose beads (Roche Diagnostics) that were pre-blocked with 2% non-fat milk powder in PBS. The proteins were eluted by incubating the beads in NuPAGE LDS sample buffer (Invitrogen) for 15 min at 37 °C. Subsequently, the eluted protein complexes were separated on a 4–12% Bis-Tris SDS-PAGE gel (Invitrogen) and transferred to a polyvinylidine difluoride (PVDF) membrane (GE Healthcare, Buckinghamshire, UK). The blot was blocked with 5% non-fat milk powder in PBS. Immunoprecipitated proteins were detected by incubation of the blots with anti-HA IgG (Roche Diagnostics) followed by incubation with anti-rat IgG conjugated to horseradish peroxidase (GE Healthcare) and subsequent ECL detection (GE Healthcare).
7
Western Blot Protein Detection
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Detection of proteins by western blot analysis was achieved using standard protocols with primary antibodies (Supplementary Table 4 ). Samples were separated on 4–12% Bis-Tris SDS–PAGE gel (Invitrogen) and transferred to polyvinylidene difluoride membrane. The membranes were then blocked in 5% milk and incubated with primary antibody in PBST overnight at 4 °C. Following incubation with the primary antibody, membranes were washed three times in PBST, incubated with IRDye (LI-COR Biosciences) secondary antibodies for 3 h, washed three times in PBST with a final PBS wash, and then visualized by the LI-COR Odyssey Imaging System (LI-COR Biosciences).
8
Western Blot Analysis of Cellular Proteins
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Cells were lysed with 9 M urea lysis buffer (20 mM HEPES, pH 8.0, 9 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, and 1 mM β-glycerophosphate) and protein amount were quantitated using Pierce™ 660nm Protein Assay Kit. Samples were then run on 4 - 12% Bis-Tris SDS-PAGE gel (Invitrogen) and transferred to a 0.2 µM PVDF membrane (Bio-Rad Laboratories). The membrane was blocked with 5% non-fat dried milk in TBST (20mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 1 h before incubating with the respective primary antibody in TBST overnight at 4 °C on a roller. Subsequently, the membrane was subjected to washing with TBST for 5 min thrice; incubation with the respective secondary antibody in 5% non-fat dried milk in TBST for 1 h; washing with TBST for 5 min thrice; 1 min incubation with the ECL substrate (Immobilon Crescendo Western HRP substrate, MerckMillipore) and exposed to X-ray film for signal detection. AXL antibody was purchased from R&D systems (AF154) and GAPDH antibody from Santa Cruz Biotechnology (sc-32233). For vitreous analysis, soluble sAPPα fragment detecting antibody was purchased from IBL (11088).
9
Quantification of Recombinant Salmon and Trout SAA
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Recombinant Atlantic salmon SAA protein (10 or 40 ng), recombinant rainbow trout SAA protein (40 ng), and supernatants (20 μL) from IL-1β-stimulated or control RTS-11 cells were used and were mixed with NuPAGE LDS loading buffer (Invitrogen, Waltham, MA, USA) containing 5% β-mercaptoethanol (β-ME, Sigma-Aldrich, St. Louis, MO, USA), incubated at 70 °C for 10 min, size separated in a 4–12% Bis-tris SDS-PAGE gel (Invitrogen, Waltham, MA, USA), and transferred to PVDF membrane (Amersham™, Merck, Rahway, NJ, USA). SAA protein was detected using the newly developed anti-SAA antibody and an anti-mouse-HRP-conjugated antibody (Sigma-Aldrich, St. Louis, MO, USA). The signal was revealed using ECL substrate (Biorad, Hercules, CA, USA) with GenoPlex system (VWR, Radnor, PA, USA) and quantified using Fiji v2.9.0 (NIH, Bethesda, MD, USA).
10
Nanobody Interaction with VAR2CSA Protein
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Recombinant full-length VAR2CSA protein was loaded (1 µg per lane) on a 4–12% Bis-Tris SDS-PAGE gel (Invitrogen) under reducing and non-reducing conditions. Proteins from the gel were transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked with a casein-containing buffer (Qiagen, Cat.no.34660) for 1 h at RT. The nanobodies (0.5 µg/ml) were added to the blocking buffer and incubated for 1 h at RT. The membrane was washed twice with TBS 0.5% Tween20 (TBST) for 10 min. HRP-conjugated goat anti-llama IgG was added (1:2000, diluted in blocking buffer) to the membrane and incubated for 1 h at RT. The membrane was washed twice with TBST and once with TBS for 10 min before detection with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).
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