The precipitated protein pellets were re-dissolved in 50 μL Laemmli buffer containing 10 mM Dithiothreitol (DTT) as a reducing agent. Samples were heated to 56°C for one hour followed by alkylation with 1% acrylamide at room temperature for 30 minutes. Sample (20 μL) was loaded onto a 15-well 4–12% NUPAGE Bis-Tris SDS PAGE gel (Thermo Fisher, Carlsbad, Calif.) and ran at 200V for 45 minutes using 3-(N-Morpholino) propane sulfonic acid (MOPS) MOPS-SDS running buffer. Resulting gels were stained with SimplyBlue Coomassie stain (Thermo Fisher) for 3 hours, de-stained in deionized water overnight, and imaged.
Nupage bis tris sds page gel
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, Canada
NuPAGE Bis-Tris SDS-PAGE gels are polyacrylamide gels designed for the separation and analysis of proteins using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique. The gels are pre-cast and contain a Bis-Tris buffer system, which provides a more stable pH environment during electrophoresis.
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Lab products found in correlation
Amersham ecl prime western blotting detection system, by GE Healthcare (3 mentions)
Immobilon p membrane, by Merck Group (3 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Chemiluminescent detection reagent, by GE Healthcare (2 mentions)
Iblot 2 gel transfer device, by Thermo Fisher Scientific (2 mentions)
Ibright fl1500, by Thermo Fisher Scientific (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Simplyblue coomassie stain, by Thermo Fisher Scientific (1 mentions)
Lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Anti β actin mab c4, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti p44 42 mapk erk1 2 ab, by Cell Signaling Technology (1 mentions)
Rabbit anti phospho stat1 ab, by Cell Signaling Technology (1 mentions)
Invitrogen ibright imaging systems 1500, by Thermo Fisher Scientific (1 mentions)
Rabbit anti stat1 ab, by Cell Signaling Technology (1 mentions)
Nupage sample buffer, by Thermo Fisher Scientific (1 mentions)
Anti β actin mab, by Santa Cruz Biotechnology (1 mentions)
Peroxidase conjugated secondary antibody, by Cytiva (1 mentions)
Enhanced chemiluminescence ecl system, by Cytiva (1 mentions)
Xcell surelock mini cell electrophoresis system, by Thermo Fisher Scientific (1 mentions)
Iblot, by Thermo Fisher Scientific (1 mentions)
11 protocols using nupage bis tris sds page gel
1
Protein Sample Preparation for SDS-PAGE
2
Ligand Blotting of Influenza Proteins
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Ligand blotting was performed using biotinylated SP-A (bSP-A) on Western blots containing electrophoretically transferred protein as described in detail previously (38 (link)). Briefly, SP-A was biotinylated in MES buffer, pH 6.0 and used for blotting at 1.3 μg/mL in TBS-T buffer (25 mM Tris, pH 7.6, 0.146 M NaCl, 0.1% Tween-20 and 5% BSA). Bound bSP-A was visualized by sequential incubation of washed blots with horseradish peroxidase (HRP)-conjugated Streptavidin and 3,3’-diaminobenzidine as the HRP substrate. Blots were developed using enhanced chemiluminescence. For blotting, purified baculovirus expressed HA or NA were obtained from BEI Resources with cat no NR-34587 (A/Toulon/1173/2011 (H1N1)pdm09 HA), NR-19240 (A/Puerto Rico/8/1934 (H1N1) HA), NR-19241 (A/New York/55/2004 (H3N2) HA), NR-2633 (A/Netherlands/219/2003 (H7N7), NR-41792 (A/Hong Kong/33982/2009 (H9N2) HA), NR-43739 (A/duck/Hunan/795/2002 (H5N1) HA), and NR-42002 (A/Puerto Rico/8/1934 (H1N1) NA). For Western blotting, 0.1 μg of recombinant protein in reducing NuPAGE LDS sample buffer (ThermoFisher, Cat No NP008) were loaded onto a 10-well 4-12% NuPAGE Bis-Tris SDS-PAGE gel (ThermoFisher, Cat No NP0322BOX), electrophoretically separated and blotted onto Nitrocellulose. The PageRuler Plus Prestain Protein Ladder (ThermoFisher, Cat No 2669) was used as molecular weight standard.
3
PD-L1 Western Blot Analysis
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Tumor cell lines (1 × 106) were washed in phosphate-buffered saline (PBS) and lysed in LDS sample buffer (Invitrogen, Thermo Fisher Scientific). The cell lysate was subjected to electrophoresis in a 4–12% NuPAGE Bis–Tris SDS-PAGE gel (Invitrogen, Thermo Fisher Scientific) under reducing condition and transferred to an Immobilon-P membrane (Merck Millipore). The membrane was blocked in PBS containing 0.01% Tween 20 and 5% non-fat dry milk for 1 h at room temperature and incubated with polyclonal rabbit anti-human PD-L1 (E1L3N, Cell Signaling Technology) diluted 1:1000 in blocking buffer for overnight at 4 °C, or anti-β-actin mAb (C4, Santa Cruz Biotechnology) diluted 1:2000 in blocking buffer as the control for 1 h at room temperature. After washing, the membrane was incubated with horseradish peroxidase-labeled sheep anti-rabbit or anti-mouse IgG and visualized using the Amersham ECL Prime Western Blotting Detection System (GE Healthcare Life Sciences).
4
Western Blot Analysis of PLAC1 Protein
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Tumor cell lines (2 × 106 cells) were washed in phosphate buffered saline (PBS). Cell lysates were extracted using a Total Protein Extraction Kit for Animal Cultured Cells and Tissues (Invent Biotechnologies, Inc., Plymouth, MN), subjected to electrophoresis in a 4–12% NuPAGE Bis-Tris SDS-PAGE gel (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA) and transferred to an Immunobilon-P membrane (Merck Millipore, Burlington, MA), followed by blocking of the membrane using PBS with 0.01% Tween 20 and 5% nonfat dry milk at room temperature. After 1 h, the membrane was incubated with anti-PLAC1 rabbit polyclonal Ab (Abgent, San Diego, CA) diluted 1:1000 in blocking buffer at 4°C overnight or anti-β-actin mouse mAb (C4, Santa Cruz Biotechnology) diluted 1:3000 in blocking buffer as the internal control for 2 h at room temperature. After washing, the membrane was incubated with horseradish peroxidase-labeled sheep anti-rabbit or anti-mouse IgG and visualized using an Amersham ECL Prime Western Blotting Detection System (GE Healthcare Life Sciences, Logan, UT).
5
Protein Extraction and Western Blot Analysis of HNSCC Cell Lines
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The total protein extraction kit for cultured animal cells and tissues (Invent Biotechnologies, Inc.) was used for protein extraction from HNSCC cell lines. Protein extracts were electrophoresed using a 4%–12% NuPAGE Bis‐Tris SDS‐PAGE gel (Invitrogen, Thermo Fisher Scientific Inc.) and transferred onto an Immobilon‐P membrane (Merck Millipore).
The membrane was then incubated with mouse anti‐human HOXB7 Ab (40–2000, 1:100 dilution, Invitrogen), rabbit anti‐phospho‐p44/42 MAPK (pERK1/2) Ab (Thr202/Tyr204; Cell Signaling Technology), rabbit anti‐p44/42 MAPK (ERK1/2) Ab (137F5; Cell Signaling Technology), mouse anti‐CIITA Ab (7‐1H; Santa Cruz Biotechnology), rabbit anti‐phospho‐Stat1 Ab (D4A7; Cell Signaling Technology), rabbit anti‐Stat1 Ab (42H3; Cell Signaling Technology), and mouse anti‐human β‐actin Ab (C4; Santa Cruz Biotechnology). The Amersham ECL Prime Western Blotting Detection System (GE Healthcare Life Sciences) and Invitrogen iBright Imaging Systems 1500 (Invitrogen) were used for detection by chemiluminescence. ImageJ was used to analyze the expression values of the target bands.25
The membrane was then incubated with mouse anti‐human HOXB7 Ab (40–2000, 1:100 dilution, Invitrogen), rabbit anti‐phospho‐p44/42 MAPK (pERK1/2) Ab (Thr202/Tyr204; Cell Signaling Technology), rabbit anti‐p44/42 MAPK (ERK1/2) Ab (137F5; Cell Signaling Technology), mouse anti‐CIITA Ab (7‐1H; Santa Cruz Biotechnology), rabbit anti‐phospho‐Stat1 Ab (D4A7; Cell Signaling Technology), rabbit anti‐Stat1 Ab (42H3; Cell Signaling Technology), and mouse anti‐human β‐actin Ab (C4; Santa Cruz Biotechnology). The Amersham ECL Prime Western Blotting Detection System (GE Healthcare Life Sciences) and Invitrogen iBright Imaging Systems 1500 (Invitrogen) were used for detection by chemiluminescence. ImageJ was used to analyze the expression values of the target bands.
6
Western Blot Analysis of EGFR and HSP Proteins
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Cells (1 × 106) were washed in phosphate-buffered saline (PBS) and lysed in NuPAGE sample buffer (Invitrogen, CA). The lysates were subjected to electrophoresis (NuPAGE bis-Tris SDS-PAGE gel (Invitrogen, CA)) and transferred to Immobilon-P membrane (Millipore, Bedford, MA). The membrane was soaked in blocking buffer (PBS containing 5% non-fat dry milk and 0.01% Tween 20, 1 h) at room temperature. Blots were then incubated with polyclonal rabbit anti-human EGFR (sc-03; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), polyclonal rabbit anti-human phospho-EGFR (Tyr1068; Cell Signaling Technology, Denver, MA), polyclonal rabbit anti-human heat shock protein 70 (HSP70) (Enzo Life Sciences, Inc., Farmingdale, NY), or monoclonal rat anti-human heat shock protein 90 (HSP90) (Enzo Life Sciences) diluted 1:500 in blocking buffer, or anti β-actin mAb (Santa Cruz Biotechnology) diluted 1:1,000 in blocking buffer, for 18 h at 4°C. The membrane was incubated with HRP-labeled sheep anti rabbit or anti mouse IgG after washing, and made visible by an enhanced chemiluminescence (ECL) system (Amersham, Buckinghamshire, UK).
7
Western Blotting of HIF-1α and AMPK in HRPTECs
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Total cellular extracts and soluble nuclear extracts from HRPTECs were prepared as described previously57 (link),58 (link). Western blotting was carried out using 3–8% Novex NuPAGE Tris-acetate gels (Invitrogen) for HIF-1α and nucleoporin p62 or 4–12% NuPAGE Bis-Tris SDS-PAGE gels (Invitrogen) for p-AMPKα (Th172), AMPK and α-actinin under reducing conditions. After proteins were transferred onto a Hybond-P PVDF membrane (Amersham Biosciences Co., Piscataway, NJ, USA), the membranes were incubated with the primary antibodies (dilution 1:1000), incubated with a peroxidase-conjugated secondary antibody (dilution 1:50000) (Amersham), and visualized with an enhanced chemiluminescence (ECL) system (Amersham). Selected blots were washed and reprobed with an antibody against nucleoporin p62 for nuclear protein extracts and α-actinin for total cellular extracts to control for small variations in protein loading and transfer. Images were processed using ImageJ (U. S. National Institutes of Health, Bethesda, MD, USA) for densitometric analysis. Signal intensities in the control lanes were arbitrarily assigned a value of 1.00.
8
Immunoprecipitation and Western Blotting of Trim33 and Ctcf
9
Western Blot Analysis of Inflammatory Proteins
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Five to ten micrograms of protein from the cell lysates was run on 4 to 12% NuPAGE Bis-Tris SDS-PAGE gels (Invitrogen by ThermoFisher Scientific) and transferred to polyvinylidene difluoride membranes (ThermoFisher Scientific) using an iBlot 2 gel transfer device (Life Technologies). Membranes were blocked in 5% nonfat dairy milk and incubated with primary antibodies overnight. The primary antibodies used are rabbit-anti-mouse monoclonal total pyrin antibody (ab195975; abcam), rabbit-anti-mouse monoclonal antibody phospho-serine 205 (ab201784; abcam), rabbit-anti-mouse/human IL-1β (number 12242; Cell Signaling), and rabbit-anti-mouse/human polyclonal β-actin (number 4967; Cell Signaling). Horseradish peroxidase-conjugated anti-rabbit antibody (Jackson Laboratory) was used as a secondary antibody. Proteins were visualized using chemiluminescent detection reagent (GE Healthcare) on an iBright FL1500 (ThermoFisher Scientific).
10
Protein Analysis of Cell Lysates
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Cell lysates were run on 4–12% NuPAGE Bis-Tris SDS-PAGE gels (Invitrogen by ThermoFisher Scientific) and transferred to PVDF membranes (ThermoFisher Scientific) using an iBlot 2 Gel Transfer Device (Life Technologies). Membranes were blocked in 5% non-fat dairy milk and incubated with primary antibody overnight. The primary antibodies used were rabbit MAb for GSDMD (Abcam, ab209845), MAb for Histone 3 (Cell Signaling #96C10), Mab for Citrullinated Histone 3 (Abcam #ab5103) and rabbit polyclonal for b-actin (Cell Signaling, #4967), and polyclonal ab for ExoS (from Arne Rietsch). HRP-conjugated anti-rabbit (Jackson Immuno Research) or HRP-conjugated anti-mouse (Jackson Immuno Research) was used as a secondary antibody. Protein bands reacting with antibodies were visualized using chemiluminescent detection reagent (GE Healthcare) on an iBright FL1500 (ThermoFisher Scientific).
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