|Reactivity||Human Mouse Rat|
|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.
|Specificity||Thymidylate Synthase Rabbit Recombinant mAb detects endogenous level of total Thymidylate Synthase.|
|Background||Thymidylate synthase (TS) is a key enzyme in the synthesis of 2'-deoxythymidine-5'-monophosphate, an essential precursor for DNA biosynthesis. In addition to its role in catalysis and cellular metabolism, it also functions as an RNA binding protein. Thymidylate synthase binds with high affinity to its own mRNA resulting in translational repression. TS is involved in a complex pathway that is related to many other folate dependent enzymes; at least three pathways have been identified and completely or partially described. The first essential pathway is the TS cycle, which is devoted to the regeneration of the reduced and active folate form (methylentetrahydrofolate, mTHF), which is necessary for the reaction that leads to dTMP from dUMP. The second TS pathway is related to the feedback regulation of TS expression. The third and most recently identified pathway involving the TS enzyme is that of regulatory TS proteins or rTS.|