|Storage buffer||10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.|
|Storage||Store at –20°C.|
1. Aspirate media from cultures and Wash the cells with 1X PBS. 2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice. 3. Sonicate for 10–15 sec to complete cell lysis and shear DNA. 4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice. 5. Centrifuge for 5 min (with Microcentrifuge). 6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins). NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer. 7. Electrotransfer to nitrocellulose/PVDF membrane.
Membrane Blocking and Antibody Incubations
1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature. 2. Incubate the membrane in the blocking buffer for 1 hr at room temperature. 3. Wash three times for 5 min each with TBST.
b. Antibodies Incubation
1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C. 2. Wash three times for 5 min each with TBST. 3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature. 4. Wash three times for 5 min each with TBST. 5. Proceed with detection.
Detection of Proteins
1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST. 2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well. 3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
NOTE: Do not allow slides to dry at any time during this procedure.
1. Deparaffinize/hydrate sections:
1. Incubate sections in three washes of xylene for 5 min each.
2. Wash sections two times in dH2O for 5 min each.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
1. Wash sections in dH2O three times for 5 min each.
|Specificity||TRX1 p53 Rabbit Recombinant mAb detects endogenous levels of total TRX1.|
|Background||Thioredoxin 1 (Trx1) is a conserved antioxidant protein that is well known for its disulfide reductase activity. It is found primarily in the cytosol and occasionally in the nucleus. TRX1 plays critical roles in regulating protein thiol homeostasis and redox signaling both inside and outside the cellular environment. Trx1 regulates a wide range of cellular functions, including cell growth, proliferation, and apoptosis. Its dysfunction is associated with a variety of diseases in which redox imbalance has been implicated, including cancer, human immunodeficiency virus infection, neurodegenerative diseases, and cardiovascular diseases. Under physiological conditions, Trx1 maintains the redox status of proteins via its reductase or denitrosylating activities, sustained with the reducing equivalent from TrxR/NADPH. Within highly oxidative environments, Trx1 becomes S-nitrosylated and offers an alternative modality of protein regulation via transnitrosylation. Transnitrosylation may serve two purposes: to temporarily prevent proteins from acquiring irreversible oxidative modifications when cellular antioxidant systems are overwhelmed, and to serve as a signaling mechanism for altering protein function, contributing to the cellular stress response.|