Nupage 4 12 bis tris sds page gel

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The NuPAGE 4–12% Bis-Tris SDS-PAGE gel is a pre-cast polyacrylamide gel used for the separation of proteins in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system. The gel has a concentration range of 4% to 12% and uses a Bis-Tris buffer system.

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Bis-Tris gels (1248 citations) NuPAGE (743 citations) NuPAGE gels (529 citations) SDS-PAGE (402 citations) SDS-PAGE gels (327 citations) NuPAGE Bis-Tris (95 citations) NuPAGE 4–12% Bis-Tris (72 citations) Bis-Tris SDS-PAGE (35 citations) NuPAGE SDS gel (19 citations) NuPage SDS-PAGE (16 citations)

13 protocols using nupage 4 12 bis tris sds page gel

Immunoblotting Protein Expression Analysis

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Protein
expression was confirmed by immunoblotting. The cell lysates (10 μL)
were loaded onto a NuPAGE 4–12% Bis-Tris SDS-PAGE gel (Invitrogen)
and then transferred to PVDF membranes (VWR) for 1 h at 100 millivolts.
Blots were blocked using 5% bovine serum albumin (US Biologicals)
in TBS-Tween buffer (50 mM Tris, 150 mM NaCl, 0.05% Tween 20) for
2 h at 4 °C, incubated with biotinylated anti-Maltose Binding
Protein antibodies (Vector laboratories) overnight at 4 °C, followed
by peroxidase-conjugated streptavidin (Jackson ImmunoResearch) for
2 h at 4 °C. Blots were developed with the Pierce ECL Western
Blotting Substrate Kit and chemiluminescence was measured using an
ImageQuant LAS 4000 (GE Healthsciences).

Khadria A.S., Mueller B.K., Stefely J.A., Tan C.H., Pagliarini D.J, & Senes A. (2014). A Gly-Zipper Motif Mediates Homodimerization of the Transmembrane Domain of the Mitochondrial Kinase ADCK3. Journal of the American Chemical Society, 136(40), 14068-14077.

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Confirming Membrane Protein Orientation

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To confirm proper membrane insertion and orientation of the TOXCAT and TOXGREEN constructs, overnight cultures were plated on M9 minimal medium plates containing 0.4% maltose as the only carbon source and grown at 37 °C for 72 hours.
For immunoblotting, the equivalent of 200 μL of culture media at a cell density of 1 OD600 were pelleted and chemically lysed with SoluLyse (Genlantis). 3 μL of cell lysates were mixed with 2× loading buffer and loaded onto a NuPAGE 4–12% Bis-Tris SDS-PAGE gel (Invitrogen), each construct was run in duplicate. Proteins were transferred to PVDF membranes (VWR) for 1 hour at 100 millivolts. Blots were blocked using 5% bovine serum albumin (US Biologicals) in TBS-Tween buffer (50 mM Tris, 150 mM NaCl, 0.05% Tween 20) overnight at 4 °C, incubated with goat biotinylated anti-Maltose Binding Protein antibodies (Vector labs) at 25 °C for 2 hours, followed by peroxidase-conjugated streptavidin anti-goat secondary antibodies (Jackson ImmunoResearch) at 4 °C for 2 hours. Blots were developed with the Pierce ECL Western Blotting Substrate Kit, 1 mL of ECL solution was added to the blot and incubated for 90 seconds. Chemiluminescence was measured using an ImageQuant LAS 4000 (GE Healthsciences). Immunoblots of samples used for direct comparison (Figs. 5, S3 and S4) were processed and developed in parallel.

Armstrong C.R, & Senes A. (2016). Screening for transmembrane association in divisome proteins using TOXGREEN, a high-throughput variant of the TOXCAT assay. Biochimica et biophysica acta, 1858(11), 2573-2583.

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Immunoblotting for Protein Expression

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Protein
expression was confirmed by immunoblotting. The cell lysates were
normalized by cell density and loaded onto a NuPAGE 4–12% bis-tris
SDS–PAGE gel (Invitrogen) and then transferred to PVDF membranes
(VWR) for 1 h at 100 millivolts. Blots were blocked using 5% bovine
serum albumin (US Biologicals) in TBS-Tween buffer (50 mM Tris, 150
mM NaCl, 0.05% Tween 20) for overnight at 4 °C, incubated with
goat biotinylated anti-maltose binding protein antibodies (Vector
laboratories) for 2 h at room temperature, followed by peroxidase-conjugated
streptavidin antigoat secondary antibodies (Jackson ImmunoResearch)
for 2 h at 4 °C. Blots were developed with the Pierce ECL Western
Blotting Substrate Kit; 1 mL of ECL solution was added to the blot
and incubated for 90 s. Chemiluminescence was measured using an ImageQuant
LAS 4000 (GE Healthsciences). Individual bands were quantified by
ImageJ.

Anderson S.M., Mueller B.K., Lange E.J, & Senes A. (2017). Combination of Cα–H Hydrogen Bonds and van der Waals Packing Modulates the Stability of GxxxG-Mediated Dimers in Membranes. Journal of the American Chemical Society, 139(44), 15774-15783.

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Venom Protein Characterization via SDS-PAGE

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A NuPAGE 4–12% Bis-Tris SDS-PAGE gel (1.0 mm × 10 well, Invitrogen) was loaded with 25 μg of total protein for each pooled, crude venom. Briefly, 2.5 μL of sample (10 mg/mL) were combined with 11 μL of SDS buffer solution and 5 μL NuPAGE reducing agent (Invitrogen) and then heated to 70 °C for 10 min. Samples were run at 100 V for 60 min; SeeBlue Plus2 Protein Standard 1X (Invitrogen) was used as the ladder. Gels were stained and destained with Simply Blue SafeStain (Invitrogen) according to the manufacturer’s protocol. Protein identification was based on approximate molecular weights as described in Mackessy (2008) .

Margres M.J., Walls R., Suntravat M., Lucena S., Sánchez E.E, & Rokyta D.R. (2016). Functional characterizations of venom phenotypes in the eastern diamondback rattlesnake (Crotalus adamanteus) and evidence for expression-driven divergence in toxic activities among populations. Toxicon : official journal of the International Society on Toxinology, 119, 28-38.

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Fibrinogenolytic Activity Assay of Snake Venoms

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We tested each pooled venom for fibrinogenolytic activity, a proxy for proteinase activity. Briefly, 0.005 g of fibrinogen (without plasminogen; HYPHEN BioMed) was dissolved in 1mL of water and incubated at 37 °C for 30 min. Following incubation, 20 μL of the fibrinogen solution were separately added to 10 μL of each C. adamanteus venom (1 mg/mL, 10 μg protein), 10 μL of 0.85% saline solution (negative control), and 10 μL of C. atrox venom (1 mg/mL, 10 μg protein; positive control). All samples were incubated at 37 °C for 24 h. Next, 11 μL of SDS buffer solution and 5 μL NuPAGE reducing agent (Invitrogen) were added and each sample was heated to 70 °C for 10 min. Fifteen μL of the sample mixture were run on a NuPAGE 4–12% Bis-Tris SDS-PAGE gel (1.0 mm × 10 well, Invitrogen) at 200 V for 35 min; SeeBlue Plus2 Protein Standard 1X (Invitrogen) was used as the ladder. Gels were stained and destained with Simply Blue SafeStain (Invitrogen) according to the manufacturer’s protocol. Absence of the α, β, and/or γ chain(s) indicated hydrolysis of that chain and was recorded as a positive result (i.e., fibrinogenolytic activity).

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Mass Spectrometry-Based Protein Identification

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Immunoprecipitation was performed using protein A Dynabeads (Life Technologies) coated with LILR-Fc protein. 50μl PBS-T-washed Dynabeads were loaded with LILR-Fc following incubation with 50μl of 250μg/ml freshly made LILR-Fc at 4°C for 1 hour followed by two washes with PBS-T. The construction and preparation of the LILR-Fc protein is described in the Supplementary Material.
Cell lysates (100μl) were precleared by incubation at 4°C for 1 hour with 25μl protein A Dynabeads. 50μl of LILR-Fc loaded Dynabeads were then added to the precleared lysate for 1hr at 4°C. Beads were washed thrice in cold PBS-T and resuspended with LDS loading buffer (30mM Glycine pH2.8, 1x Nupage LDS buffer and reducing agent [Life Technologies]). Samples were denatured at 70°C for 10 min, before loading onto a Nupage 4-12% Bis-Tris SDS PAGE gel (Life Technologies) alongside SeeBlue II marker (Life Technologies). Gels were run in MOPs buffer (Life Technologies) for 45 minutes at 200V. Protein bands were visualised following Coomassie Blue staining.
Protein bands in gels were identified by fingerprinting of tryptic peptide using an Applied Biosystems 4800 Plus MALDI-TOF-TOF Mass Spectrometer in CIMR/IMS Proteomics Facility (CIPF) of the Cambridge Institute for Medical Research.

Jones D.C., Hewitt C.R., López-Álvarez M.R., Jahnke M., Russell A.I., Radjabova V., Trowsdale A.R, & Trowsdale J. (2016). Allele-specific recognition by LILRB3 and LILRA6 of a cytokeratin 8 - associated ligand on necrotic glandular epithelial cells. Oncotarget, 7(13), 15618-15631.

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SDS-PAGE Analysis of rVAR2-SpyC Conjugation

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The rVAR2 proteins were tested by SDS-PAGE to confirm purity and SpyC conjugation ability. rVAR2 was mixed with SpyC in a 1:1 ratio for 30 min. The mixture was then diluted in DPBS to reach a rVAR2 concentration of 0.1 µg/µL. 12.5 µL rVAR2-SpyC conjugation mixture was prepared with 2.5 µL SDS (+/− DTT) solution. The samples were placed in a 95 °C heating block for 10 min prior to running them on a NuPAGE 4–12% Bis-Tris SDS-PAGE gel (Life technologies, NP0321PK2) with a PageRuler^TM Plus prestained protein ladder (10–250 kDa) (26619, Thermo Fisher Scientific) as a size reference. The gel was run at 170 V for 1 h and was subsequently stained with InstantBlue Coomassie Protein Stain (ISB1L, Expedeon, Cambridge, UK) for at least 1 h. The gel was destained in deionized water for 1 h prior to imaging.

Sand N.T., Petersen T.B., Bang-Christensen S.R., Ahrens T.D., Løppke C., Jørgensen A.M., Gustavsson T., Choudhary S., Theander T.G., Salanti A, & Agerbæk M.Ø. (2020). Optimization of rVAR2-Based Isolation of Cancer Cells in Blood for Building a Robust Assay for Clinical Detection of Circulating Tumor Cells. International Journal of Molecular Sciences, 21(7), 2401.

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Western Blot Analysis of Mitochondrial Proteins

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For Western blot analysis, whole-cell homogenates were used. Harvested cells were resuspended in cold RIPA buffer (Thermo Fisher Scientific) supplemented with protease inhibitors cOmplete™ Protease Inhibitor Cocktail (Roche) and phosphatase inhibitors PhosSTOP EASYpack phosphatase inhibitors (Roche). Protein concentrations were assessed using a BCA Protein Assay Kit (Thermo Fisher Scientific), and 10 µg of total protein lysates were loaded per well on a NuPAGE 4–12% Bis-Tris SDS-PAGE gel (Thermo Fisher Scientific). After electrophoresis, proteins were transferred onto a PVDF membrane (BioRad) and probed with antibodies raised against MFN2 (Abcam, ab56889), TOM70 (Abcam, ab135602), SOD2 (Cell Signaling, 13141 S), Parkin (Abcam, 77924), and GAPDH (Millipore, MAB374). Subsequently, the blots were incubated with the corresponding secondary antibodies, and target proteins were detected by enhanced chemiluminescence using Clarity™ ECL Western Kit (BioRad). The chemiluminescence signal was detected using the ChemiDoc™ Touch Imaging System (BioRad) and quantified by densitometry using Image Lab 6.0 analyzer software (BioRad). Optical density values assessed for target proteins were normalized by the indicated loading control. For each figure, all blots were processed in parallel and originated from the same experiment.

Castelo Rueda M.P., Zanon A., Gilmozzi V., Lavdas A.A., Raftopoulou A., Delcambre S., Del Greco M F., Klein C., Grünewald A., Pramstaller P.P., Hicks A.A, & Pichler I. (2023). Molecular phenotypes of mitochondrial dysfunction in clinically non-manifesting heterozygous PRKN variant carriers. NPJ Parkinson's Disease, 9, 65.

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Extraction and Characterization of Leptospiral LPS

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Leptospiral LPS was extracted from the whole-inactivated bacterial preparations mentioned above by the hot phenol/water method, as described previously (33 , 34 (link)). Then, the aqueous phase of phenol/water extractions was selected for purification of leptospiral LPS by solid phase extraction on C8 reversed-phase cartridges (34 (link)). LPS concentration was determined by analyzing the content of 3-hydroxydodecanoic acid in the samples by gas chromatography, as described earlier (35 (link)). All leptospiral LPS preparations were diluted to the lowest concentration (0.056 µmol/ml) in culture medium and 1:1000 dilution of the resulting suspensions was used to stimulate cells throughout this study unless specified otherwise. SDS-PAGE was performed to assess lipoprotein contamination of isolated leptospiral LPS. Samples were separated in a NuPAGE™ 4-12% Bis-Tris SDS-PAGE gel (Thermo Scientific) and stained with Coomassie dye R-250 using the ready-to-use colorimetric Imperial™ Protein Stain (Thermo Scientific). The Coomassie-stained gel image was digitally recorded using an Epson scanner.

Novak A., Pupo E., van’t Veld E., Rutten V.P., Broere F, & Sloots A. (2022). Activation of Canine, Mouse and Human TLR2 and TLR4 by Inactivated Leptospira Vaccine Strains. Frontiers in Immunology, 13, 823058.

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Polyacrylamide Gel Electrophoresis Protocols

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Reaction products were analyzed using polyacrylamide gel electrophoresis (PAGE). Denaturing PAGE gels (12%) were prepared by mixing 17 mL UreaGel Diluent (National Diagnostics), 17 mL UreaGel Concentrate (National Diagnostics), and 4 mL 10x Tris-Borate EDTA (TBE) (Gibco by Life Technologies), 320 μL ammonium persulfate (APS) (1%), and 16 μL tetramethylethylenediamine (TEMED), both purchased from Sigma-Aldrich. Gels were prerun for 30 min at 20 W before loading the samples. Native PAGE gels (12%) contained 12 mL AccuGel 29:1 (40%) (National Diagnostics), 2 mL 10× TBE, 26 mL MilliQ, 320 µL APS, and 16 µL TEMED. All PAGE gels were stained with 5 µL SYBR Gold (Invitrogen) in 200 mL MilliQ water for 15 min and scanned on an Amersham Typhoon 5 scanner (GE Healthcare). An Ultralow range DNA ladder (10–300 bp, Thermo Scientific) was used as a size marker. NuPAGE 4–12% Bis-Tris SDS-PAGE gel (ThermoFisher) was run in MES buffer (ThermoFisher) at 150 V for 40 min. Subsequently, the gel was stained in Coomassie Brilliant Blue for 1 h and destained in MilliQ water O/N before scanning on a GelDoc Ez Imager (Bio-Rad).

Ferapontov A., Omer M., Baudrexel I., Nielsen J.S., Dupont D.M., Juul-Madsen K., Steen P., Eklund A.S., Thiel S., Vorup-Jensen T., Jungmann R., Kjems J, & Degn S.E. (2023). Antigen footprint governs activation of the B cell receptor. Nature Communications, 14, 976.

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