|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.
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||Huntingtin Rabbit Recombinant mAb detects endogenous levels of total Huntingtin.|
|Background||Huntington's Disease (HD) is a progressive and disabling neurodegenerative disorder of the central nervous system. This disease is caused by inheritance of an autosomal dominant mutation in the Huntingtin (Htt) protein. Huntingtin (Htt) is widely expressed during development and has a complex and dynamic distribution within cells. It is predicted to be a protein of pleiotropic function, interacting with a large number of effector proteins to mediate a host of physiological processes. A large number of Htt protein interactors function in microtubule-based axon trafficking. Huntingtin-associated protein 1 (HAP1) helps mediate the interaction between Htt, microtubule motor proteins and their co-factors, including kinesin, dynactin, and dynein. Huntingtin is involved in both antero- and retrograde axon transport. Htt associates with the endocytosis proteins clathrin and dynamin, as well as endocytic organelle trafficking proteins such as Endophilin 3, α-Adaptin, HIP-14, HAP1, and Huntingtin-associated protein 40 (HAP40). Htt is also associated with the movement of mitochondria along neurites and has neuroprotective functions during numerous proapoptotic challenges.|