|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.
Specimen Preparation (forcultured cell lines, IF-IC)
1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
1. Add theblocking buffer and incubate for 60 min at RT.
|Specificity||Hsc70 Rabbit Recombinant mAb detects endogenous levels of total Hsc70.|
|Background||Hsc70, also known as Hsp73 or HSPA8 is a heat shock protein and a chaperone protein. It represents a constitutively expressed, cognate protein of the HSP70 family, which is central in many cellular processes. Hsc70 represents constitutively expressed, cognate protein of the HSP70 family. It is the main housekeeping protein of the family and acts as a clathrin-uncoating ATPase during clathrin-mediated endocytosis. HSPA8 also displays important functions in cellular protein degradation, protein folding and protein import into organelles or cellular compartments. The chaperone is able to shuttle between the nucleus and the cytoplasm in an ATP-dependent manner. This property enables HSPA8 to import different cytoplasmic proteins into the nucleus such as karyophilic proteins. Besides its ntracellular location. HSPA8 is also detected in the extracellular space. Extracellular HSPA8 inhibits tumor cell growth and modifies cell viability. HSPA8 also associates with the cell surface where it might contribute to cell recognition by immune cellor act as a cellular receptor for viral infe.|