Western Blot : 1:1,000-1:2,000
Immunofluorescence/Immunocytochemistry: 1:500-1:2,000
Immunohistochemsitry (paraffin, formalin, frozen Tissues): 1:500-1:1,000

High-mobility group proteins were named originally since they are abundant, relatively low molecular weight and exhibit “high mobility”, in other words, they run quickly on SDS-PAGE gels. High-mobility group proteins box 1 (HMGB1) is one of these. The “box” in the name refers to the so-called high mobility group box, a compact domain involved in DNA binding and protein-protein interactions. The HMGB1 molecule has two of these HMG domains (1). The protein is also called amphoterin, this name being derived from the presence of two highly charged regions in the molecule, a relatively neutrally charged N-terminus and a very negatively charged C-terminus. The molecule is very unusually charged throughout, the human sequence consisting of 16.7% Glutamic acid, 9.3% Aspartic acid, 20% Lysine and 9.3% Arginine. HMGB1 can bind toll like receptor 2, 4 and 9 (TLR2, TLR4 and TLR9) and the receptor for advanced glycation end products (RAGE), (3,4). TLRs are components of the innate immune system, first recognized as a family of receptors which recognize “pathogen associated molecular pattern” molecules or (PAMPs). These are common components of bacteria and when TLRs bind these, a strong inflammatory response is activated. More recently it has been recognized that TLRs can also be activated by “damage associated molecular pattern” molecules or (DAMPs), substances released from damaged and diseased cells which also bind to TLR family receptors and also activate inflammation. HMGB1 is such a DAMP, binding to TLR4, and much evidence suggests that HMGB1 is a strong activator of inflammation. Interestingly, HMGB1 is released by necrotic cells but not by apoptotic cells