|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||TRK fused gene Rabbit Recombinant mAb detects endogenous level of total TRK fused gene.|
|Background||Trk-fused gene (TFG) is was initially identified as an oncogene causing thyroid cancer, in which the N-terminal half of TFG was fused with neurotrophic tyrosine kinase receptor 1 (NTRK1, also known as TrkA). Subsequently, TFG was also reported to be a fusion partner of the anaplastic lymphoma kinase (ALK) gene in anaplastic large cell lymphoma, though the function of TFG itself remained essentially unknown until quite recently. It was reported localized at endoplasmic reticulum (ER) exit sites (ERES) and to be crucial in transport from the ER to the Golgi apparatus via COPII vesicle. TFG is important for retention of COPII vesicles between the ER and ER-Golgi intermediate compartments (ERGIC), and in the absence of TFG, COPII-coated carriers become dispersed throughout the cytoplasm. On the other hand, TFG was recently identified as a causative gene for several neurodegenerative diseases, such as hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P), the axonal type of Charcot-Marie-Tooth disease and hereditary spastic paraplegia (HSP).|