|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.
TNF Receptor II Antibody detects endogenous levels of total TNF Receptor II.
Tumor necrosis factor (TNF) is a pleiotropic cytokine involved in regulating diverse bodily functions including cell growth modulation, inflammation, tumorigenesis, viral replication, septic shock, and autoimmunity. TNF exerts its effects through two receptors: TNFR1 and TNFR2. They possess different patterns of expression. TNFR1 is ubiquitously expressed on the lymphoid system and nearly all cells of the body, which likely accounts for TNF's wide-ranging functions. TNFR2 is expressed in a more limited manner on certain populations of lymphocytes, including T-regulatory cells (Tregs), endothelial cells, microglia, neuron subtypes, oligodendrocytes, cardiac myocytes, thymocytes, islets of Langerhans, and human mesenchymal stem cells. TNF depends on TNFR1 for apoptosis and TNFR2 for any function related to cell survival, although there is some degree of overlapping function depending upon the activation state of the cell and a variety of other factors. Likewise, TNFR1 and TNFR2 have distinct intracellular signaling pathways, although there is some overlap and crosstalk. TNFR2 signaling relies on TRAF2 and activation and nuclear entry of the pro-survival transcription factor nuclear factor-kB (NFkB). TNFR1 typically signals cell death, while TNFR2 typically signals cell survival, the ratio of their co-expression will shift the balance between cellular survival and apoptosis.