|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||Hsp27 Rabbit Recombinant mAb detects endogenous levels of total Hsp27.|
|Background||Heat shock protein 27 (HSP27) belongs to the small molecular weight heat shock protein (HSP) family (12–43 kDa). HSP27 is a multidimensional protein which acts as a protein chaperone and an antioxidant and plays a role in the inhibition of apoptosis and actin cytoskeletal remodeling. During oxidative stress, HSP27 functions as an antioxidant, lowering the levels of reactive oxygen species (ROS) by raising levels of intracellular glutathione and lowering the levels of intracellular iron. The protein functions as an anti-apoptotic agent under conditions of chemical stress by interacting with both mitochondrial dependent and independent pathways of apoptosis. HSP27 binds DAXX during Fas-FasL mediated apoptosis and prevents the subsequent binding of Ask1 by DAXX. HSP27 also interacts with Bax and cytochrome c, thereby preventing mitochondrial dependent apoptosis. HSP27 is particularly involved in protection from programmed cell death by inhibition of caspase-dependent apoptosis. HSP27 has been characterized with the ability to regulate actin cytoskeletal dynamics during heat shock and other stress conditions, functioning both to promote actin polymerization and as an actin capping protein. HSP27 is present at basal levels in cells and tissues, and is organized as large oligomers. The protein is phosphorylated by MAPKAP kinase 2/3 via the activation of the P38 MAPK pathway at multiple serine residues (15, 78, and 82 in humans and 15 and 86 in rodent HSP25). Following phosphorylation, HSP27 reorganizes itself into smaller oligomers, often dimers and tetramers and can interact with other proteins. HSP27 phosphorylation is dynamic and regulated by cellular conditions.|