Sub-confluent cells were treated with 0, 2.5 and 5μM of OSU-T315 for 48 and 72 hours for cell lines, and 72 hours for primary cells. Cells were then harvested and homogenized in lysis buffer (1% SDS, 10mM EDTA, 50mM Tris, pH 8.1) supplemented with protease/phosphatase inhibitor cocktail (Thermo Scientific, Waltham MA). Electrophoresis was performed with 25μg or 50μg of protein/lane in a 4-20% gradient SDS- polyacrylamide gel (Thermo Scientific, Waltham MA). Proteins were transferred to PVDF membranes (Millipore, Billerica MA), and probed with specific antibodies of interest and horseradish peroxidase-conjugated secondary antibodies. The chemiluminescent signals were detected in X-ray films.
Gradient sds polyacrylamide gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 4-12% gradient SDS-polyacrylamide gel is a laboratory equipment used for separating proteins based on their molecular weight. It is a pre-cast gel that creates a gradual increase in pore size from the top to the bottom of the gel, allowing for efficient separation of a wide range of protein sizes.
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Lab products found in correlation
Pvdf membrane, by Merck Group (7 mentions)
Lids loading sample buffer, by Thermo Fisher Scientific (4 mentions)
Nupage lds loading buffer, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by GE Healthcare (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Hrp conjugated streptavidin, by Thermo Fisher Scientific (3 mentions)
Ecl reagent, by Thermo Fisher Scientific (3 mentions)
Gapdh, by Abcam (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Enhanced chemiluminescence reagent, by Cytiva (2 mentions)
β dystroglycan β dg, by Santa Cruz Biotechnology (2 mentions)
Caveolin 3, by BD (2 mentions)
Dystrophin, by Abcam (2 mentions)
Dysferlin, by Abcam (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Na934, by GE Healthcare (2 mentions)
Ecl kit, by GE Healthcare (2 mentions)
Ab75853, by Abcam (2 mentions)
Amersham hyperfilm ecl, by GE Healthcare (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
31 protocols using gradient sds polyacrylamide gel
1
Dose-Dependent Protein Analysis of OSU-T315
2
Western Blot Analysis of Protein Expression
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Samples were separated on 16% SDS polyacrylamide gel or pre-made 4–20% gradient SDS polyacrylamide gel (ThermoFisher) and transferred to PVDF membranes (GE Healthcare). The membranes were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde for 30 min at room temperature and probed with various antibodies. The primary antibodies were anti-αSyn phospho (Ser129) (Abcam 51,253, 1:2500), anti-αSyn (BD Biosciences clone 42, 1:2500), anti-ATPIF1 (ThermoFisher, 1:1500), anti-ATP5A (Abcam, 1:1000), anti-GAPDH (Stressgen, 1:1000), anti-Calnexin (Sressgen, 1:1000), and anti-Syntaxin 6 (Sigma, 1:1000). The HRP-conjugated goat anti-mouse or anti-rabbit IgG antibodies (Bio-Rad) were used as the secondary antibodies. Signals were developed by ECL 2 substrate (Pierce) and scanned with ChemiDoc (Bio-Rad). The signal intensity was measured with the FIJI software.
3
Quantification of Viral-Induced PD-L1 Expression
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A total of 5 × 106 cells cultured in six-well plates were infected with indicated VVs at MOI = 2. After incubation for 48 h, supernatants were harvested and clarified by centrifugation at 10,000 × g for 2 min. Cells were lysed in 1× RIPA buffer (Sigma-Aldrich, St Louis, MO) and 1× mammalian protease inhibitor (Sigma-Aldrich, St Louis, MO) for 15 min on ice and clarified by centrifugation at 10,000 × g for 2 min. Samples of both supernatants and cell lysates were mixed with 6× sodium dodecyl sulfate (SDS) sample buffer (Bioworld, Dublin, OH) and electrophoresed in a 4–20% gradient SDS–polyacrylamide gel (Thermo, Waltham, MA). The fractionated protein samples are transferred onto 0.2 μm nitrocellulose membrane (Thermo, Waltham, MA). The nitrocellulose membrane is blocked for 30–60 min at room temperature (RT) in TBS buffer (Bio-Rad, Irvine, CA) containing 5% nonfat milk. Immunodetection of iPDL1 is performed by incubation with RD800-conjugated goat anti-mouse IgG antibody (Licor, Lincoln, NE) at RT for 1 h, or with rat anti-mouse PD-1 (Biolegend, San Diego, CA) at 1 µg/mL for overnight at 4 °C followed by with an RD800-conjugated anti-Rat IgG (Licor, Lincoln, NE). The blots are detected with an Odyssey Imager (LI-CON, Lincoln, NE).
4
Immunoblotting Analysis of NFX1 Isoforms
5
Western Blot Analysis of Protein Complexes
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HEK293T cells were lysed with RIPA buffer. Supernatants after centrifugation (16,000g, 30 min, 4 °C) were resuspended in LiDS loading sample buffer (Thermo Fisher Scientific) containing 2-mercaptethanol. The protein samples were loaded onto a 4–12% gradient SDS–polyacrylamide gel (Thermo Fisher Scientific) and separated using electrophoresis. The proteins were then transferred to a PVDF membrane (Merck Millipore) using a semidry western blot transfer system set to a constant current of 200 mA for 30 min and stained with Ponceau S (Beacle). The membranes were blocked by incubation in 5% (w/v) BSA in Tris-buffered saline and 0.1% Tween (TBS-Tween) and then incubated for 1 h or overnight. Membranes were washed three times in TBS-Tween and developed with ECL reagent (Thermo Fisher Scientific). The primary antibodies for Flag tag, PKA, CK2 were purchased from Cell Signaling Technology. The HRP-conjugated streptavidin was purchased from Thermo Fisher Scientific.
6
Western Blot Analysis of Protein Expression
7
Biotinylation and Western Blot Analysis
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HeLa cells were lysed with lysis buffer (100 mM HEPES-NaOH pH 7.5, 1% NP-40, 150 mM NaCl and 1% protease inhibitor). Supernatants after centrifugation (16,000 g, 30 min, 4 o C) were biotinylated by a click reaction as described above. The samples were re-suspended in LiDS loading sample buffer (Invitrogen) with 50 mM DTT and incubated at 70 o C for 5 min. The protein samples were loaded onto a 4-12% gradient SDS-polyacrylamide gel (Thermo Fisher Scientific) and separated using electrophoresis. The proteins were then transferred to a PVDF membrane (Merck Millipore) using a semi-dry western blot transfer system set to a constant current of 200 mA for 30 min. The membranes were first blocked by incubating in 5% (w/v) BSA in Tris-buffered saline and 0.1% tween (TBS-tween) and then incubated with HRP-conjugated Streptavidin (Thermo Fisher Scientific) diluted 1:50,000 in 5% BSA in TBS-tween for 4 h while rotating at room temperature. Membranes were washed three times in TBS-tween and developed with ECL reagent (Thermo Fisher Scientific).
8
Western Blot Protein Analysis Protocol
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The cell lysates were re-suspended in LiDS loading sample buffer (Thermo Fisher Scientific) with 50 mM DTT and incubated at 70°C for 5 min. The protein samples were loaded onto a 4–12% gradient SDS-polyacrylamide gel (Thermo Fisher Scientific) and separated using electrophoresis. The proteins were then transferred to a PVDF membrane (Merck Millipore) using a semi-dry western blot transfer system set to a constant current of 200 mA for 30 min. The membranes were first blocked by incubation in 5% (w/v) BSA or 5% (w/v) skim milk in Tris-buffered saline and 0.1% tween 20 (TBS-T) and then incubated with anti-puromycin antibody (clone 3RH11, Cosmo Bio) diluted 1:5,000, overnight at room temperature. The membrane was washed three times with 0.1% TBS-T, incubated with HRP-conjugated anti-mouse secondary antibody (1: 20,000 dilution) in 0.1% TBS-T 1 h at room temperature, washed three times in TBS-T, and developed with ECL reagent (Cytiva). Ubiquitinated proteins were detected using anti-ubiquitin rabbit antibody (CST) (1: 2,000 dilution) and HRP-conjugated anti-rabbit secondary antibody (1: 10,000 dilution). Chemiluminescence was detected using the luminescent image analyzer ImageQuant LAS-500 (Cytiva).
9
Biotinylation and Western Blot Analysis
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HeLa cells were lysed with lysis buffer (100 mM HEPES-NaOH pH 7.5, 1% NP-40, 150 mM NaCl and 1% protease inhibitor). Supernatants after centrifugation (16,000 g, 30 min, 4 o C) were biotinylated by a click reaction as described above. The samples were re-suspended in LiDS loading sample buffer (Invitrogen) with 50 mM DTT and incubated at 70 o C for 5 min. The protein samples were loaded onto a 4-12% gradient SDS-polyacrylamide gel (Thermo Fisher Scientific) and separated using electrophoresis. The proteins were then transferred to a PVDF membrane (Merck Millipore) using a semi-dry western blot transfer system set to a constant current of 200 mA for 30 min. The membranes were first blocked by incubating in 5% (w/v) BSA in Tris-buffered saline and 0.1% tween (TBS-tween) and then incubated with HRP-conjugated Streptavidin (Thermo Fisher Scientific) diluted 1:50,000 in 5% BSA in TBS-tween for 4 h while rotating at room temperature. Membranes were washed three times in TBS-tween and developed with ECL reagent (Thermo Fisher Scientific).
10
Biotinylated ssDNA Affinity Purification
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Custom 5′-terminal biotinylated single-strand (ss) DNAs corresponding to 3′UTRs from ζWT (5′TGGAGGTTCCCCAGCCCCACTTACCGCGTAAT3′), ζ57 (5′TGGAG GTTAGTGCACACCACTTACCGCGTAAT3′), and ζ65 (5′TGGAGGTTCCCCAGCCAGTGCACACGCGTAAT3′) mRNAs were commercially sourced (IDT). Approximately 1 μg of each ssDNA was incubated overnight at 4°C in 500 μL cytoplasmic extract supplemented with 50 μL ImmunoPure Avidin Agarose beads (Pierce). The pelleted beads were washed 2× with PBS + Triton-×100 (0.05%) and 2× with PBS + Triton-×100 (1.0%), resuspended in 10 μL loading buffer, and resolved on a precast 4-12% gradient SDS-polyacrylamide gel (Invitrogen). Cytoplasmic extracts were prepared from ~1×107 cells lysed in buffer (1 mM Hepes pH 7.9, 0.15 mM MgCl2, 1 mM KCl) and clarified by centrifugation at 13000xg for five minutes at 4°C; extract was stored in aliquots at −80°C.
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