|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||Osteopontin Rabbit Recombinant mAb detects endogenous level of total Osteopontin.|
|Background||Osteopontin (OPN) is a secreted matricellular protein involved in several physiological as well as pathological events. It can also be found in the cytoplasm and the nucleus suggesting that the intracellular form (iOPN) may be involved in alternative translation and has biological functions distinct from those of secreted OPN (sOPN). OPN is expressed in various cell types and tissues including preosteoblasts, osteoblasts, osteocytes, chondrocytes, fibroblasts, dendritic cells, macrophages and T-cells, hepatocytes, smooth muscle cells, skeletal muscle, endothelial cells, inner ear, brain, placenta and mammary glands, deciduum and kidney. Extracellular OPN functions through its interactions with multiple cell surface receptors, including various integrins (αvβ1, αvβ3, αvβ5, αvβ6, α4β1, α5β1, α8β1, and α9β1) and CD44 thereby regulating cellular processes such as biomineralization, tissue remodeling and immune regulation. Osteopontin plays a critical role in chronic inflammatory diseases, including multiple sclerosis, Crohn's disease and other autoimmune disorders, several types of cancer and cardiovascular diseases.|