Protein analysis was performed via western blotting on tissue homogenates in passive lysis buffer (Promega), separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane (Whatman) and blotted for CGL (ab151769 Abcam), CBS (ab135626 Abcam), MST (#85211 Abcam), or α-Tubulin (#4074 Abcam), followed by horseradish peroxidase-conjugated secondary anti-rabbit antibody (#97051 Abcam). Proteins were visualized using SuperSignal West Femto Maximum Sensitivty Substrate (Thermo Scientific #34096) on an Amersham Imager 600 (General Electric) and sizes determined using the PageRuler Plus Prestained (26619 Thermo Fisher). After blotting and washing, the membranes were stained using Coomassie blue, to determine the relatively equal amount of tissue proteins loaded.
Pageruler plus prestained
Manufactured by Thermo Fisher Scientific
The PageRuler Plus Prestained Protein Ladder is a ready-to-use protein standard for monitoring protein separation and estimating the molecular weight of proteins during SDS-PAGE and western blotting. It contains pre-stained proteins with a molecular weight range from 10 to 250 kDa.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Cytiva (1 mentions)
Ab151769, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody, by Abcam (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Coomassie brilliant blue g 250, by Merck Group (1 mentions)
Nu page lithium dodecyl sulfate sample buffer, by Thermo Fisher Scientific (1 mentions)
Methanol ar grade, by Merck Group (1 mentions)
Lysozyme from chicken egg white, by Merck Group (1 mentions)
3 protocols using pageruler plus prestained
1
Western Blot Analysis of Sulfur Metabolism
2
SDS-PAGE Protein Separation Protocol
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Samples were mixed with 4× SDS-PAGE loading buffer (200 mM Tris-Cl, pH 6.8, 400 mM DTT, 8% SDS, 0.4% bromophenol blue, 40% glycerol), boiled at 95 °C for 10 min and loaded onto a 1 mm thick SDS-PAGE gel containing 16% acrylamide. The gels were run at 100 V and stained with coomassie Brilliant Blue G-250. PageRuler (Plus) Prestained (Thermo Fisher Scientific) was used as molecular weight reference marker.
3
Protein Analysis Protocol Using LDS-PAGE
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Benzene (AR grade), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES), sodium dodecylbenzenesulfonate (DBS), and Coomassie
Brilliant Blue G-250 were purchased from Sigma-Aldrich (St. Louis).
Methanol (AR grade) and lysozyme (from chicken egg white, protein
≥90%, ≥40 000 units/mg protein, stored at −20
°C) were purchased from Merck (Kenilworth). Poly(styrene-block-ethylene oxide) (PS-b-PEO, 25.5 kg/mol, Đ = 1.08, PS/PEO ratio of 71/29) was purchased from
Polymer Source Inc. (Montreal, Canada). mPEG-succinimidyl (mPEG-SCM,
2 kg/mol) was purchased from JenKem Technology (Plano). Float-A-Lyzer
G2 Dialysis Device (5 mL MWCO 8–10 kD) was purchased from Lab
Supply (Dunedin, New Zealand). LDS-PAGE setup, NuPAGE materials (NuPage
lithium dodecyl sulfate sample buffer and NuPage sample reducing agent),
and PageRuler Plus Prestained were purchased from Thermo Fisher (Waltham).
The type 1/ultrapure water (18.2 MΩ·cm) was sourced from
a Milli-Q Direct 8 water purification system.
acid (HEPES), sodium dodecylbenzenesulfonate (DBS), and Coomassie
Brilliant Blue G-250 were purchased from Sigma-Aldrich (St. Louis).
Methanol (AR grade) and lysozyme (from chicken egg white, protein
≥90%, ≥40 000 units/mg protein, stored at −20
°C) were purchased from Merck (Kenilworth). Poly(styrene-block-ethylene oxide) (PS-b-PEO, 25.5 kg/mol, Đ = 1.08, PS/PEO ratio of 71/29) was purchased from
Polymer Source Inc. (Montreal, Canada). mPEG-succinimidyl (mPEG-SCM,
2 kg/mol) was purchased from JenKem Technology (Plano). Float-A-Lyzer
G2 Dialysis Device (5 mL MWCO 8–10 kD) was purchased from Lab
Supply (Dunedin, New Zealand). LDS-PAGE setup, NuPAGE materials (NuPage
lithium dodecyl sulfate sample buffer and NuPage sample reducing agent),
and PageRuler Plus Prestained were purchased from Thermo Fisher (Waltham).
The type 1/ultrapure water (18.2 MΩ·cm) was sourced from
a Milli-Q Direct 8 water purification system.
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