Thirty micrograms of different protein fractions from rat tissues were subjected to 10% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to Immobilon polyvinylidene difluoride transfer membranes (Millipore, Burlington, MA, USA) and blocked for 1 h at room temperature with 5% non-fat milk solution in 0.1% Tween-20-Tris-buffered saline (TBS). Membranes were then incubated overnight with the primary antibody in 0.1% Tween-20-TBS with 5% bovine serum albumin (BSA) at 4 °C. Detection was achieved using the enhanced chemiluminescence (ECL) kit for horseradish peroxidase (HRP) (Amersham GE Healthcare Europe GmbH, Barcelona, Spain). To confirm the uniformity of protein loading, the blots were incubated with β-actin or β-tubulin antibody (Sigma-Aldrich, Saint Louis, MO, USA) as a control. The size of the detected proteins was estimated using protein molecular-mass standards (Invitrogen, Life Technologies). Primary antibodies for phosphor- and total-IRE1, ATF6α, phosphor- and total-PERK, and XBP1s proteins were obtained from Santa Cruz Biotechnologies (Dallas, TX, USA) and AbCam (Cambridge, UK) as described previously [32 (link)].
Protein molecular mass standards
Manufactured by Thermo Fisher Scientific
Protein molecular-mass standards are a set of proteins with known molecular masses used to determine the molecular weights of unknown proteins in a sample. They are commonly used in Western blot analysis and other protein separation techniques.
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2 protocols using protein molecular mass standards
1
Western Blot Analysis of ER Stress Proteins
2
Western Blot Analysis of Nrf2 and HO-1
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Thirty micrograms of different protein fractions from rat tissues were subjected to 10% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to Immobilon polyvinylidene difluoride transfer membranes (Millipore, Billerica, MA, USA) and blocked for 1 h at room temperature with 5% non-fat milk solution in 0.1% Tween-20-Tris-buffered saline (TBS). Membranes were then incubated overnight with the primary antibody raised against Nrf2 or HO-1, in 0.1% Tween-20-TBS with 5% bovine serum albumin (BSA) at 4ºC.
Detection was achieved using the enhanced chemiluminescence (ECL) kit for horseradish peroxidase (HRP) (Amersham GE Healthcare Europe GmbH, Barcelona, Spain). To confirm the uniformity of protein loading, the blots were incubated with β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA) as a control. The size of detected proteins was estimated using protein molecular-mass standards (Invitrogen, Life Technologies). Primary antibodies for HO-1 and Nrf2 were obtained from Santa Cruz Biotechnologies (Dallas, TX, USA).
Detection was achieved using the enhanced chemiluminescence (ECL) kit for horseradish peroxidase (HRP) (Amersham GE Healthcare Europe GmbH, Barcelona, Spain). To confirm the uniformity of protein loading, the blots were incubated with β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA) as a control. The size of detected proteins was estimated using protein molecular-mass standards (Invitrogen, Life Technologies). Primary antibodies for HO-1 and Nrf2 were obtained from Santa Cruz Biotechnologies (Dallas, TX, USA).
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