|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||HMGB1 Rabbit Recombinant mAb detects endogenous levels of total HMGB1.|
|Background||High mobility group box 1 protein (HMGB1) is an evolutionarily ancient protein that was discovered recently to have a new guise as a cytokine. The early studies of HMGB1 focused on its role as a DNA-binding protein, as it was found to be co-purified with chromosomal DNA. However, it was only loosely bound to chromatin, unlike the more tightly bound histones. HMGB1 is a nuclear protein that is present in almost all eukaryotic cells, and it functions to stabilize nucleosome formation and acts as a transcription-factorlike protein that regulates the expression of several genes. It is now known that HMGB1 is also secreted by activated macrophages, mature dendritic cells (DCs), and natural killer (NK) cells in response to injury, infection or other inflammatory stimuli. HMGB1 causes DNA bending and facilitates the binding of several regulatory protein complexes to DNA, particularly members of the nuclear hormone-receptor family, V(D)J recombinases, and p53–p73 transcriptional complexes. it also facilitates the integration of transposons. HMGB1 enhances the interaction of other proteins with DNA and enhances transcriptional activation. HMGB1 mediates systemic inflammatory responses, is secreted by activated immune cells, activates prototypical inflammatory responses in immune cells and endothelial cells, and transduces cellular signals through RAGE (receptor for advanced glycation end-products) and possibly other receptors, such as Toll-like receptor 2 (TLR2) and TLR4. HMGB1 is expressed by almost all cells, excepting those that have eliminated their nucleus (such as erythrocytes and cornifying epithelial cells in the skin and proximal gut). HMGB1 occupies a central role in mediating the local and systemic responses to necrotic cell death and cancer, invasion by pathogens, trauma and sepsis. It has differential tissuespecific activities.|