Ab97083

Extracellular histone H3 levels were determined using a semi-quantitative method previously described^{50 (link),51 (link)}. Briefly, plasma dilutions were subjected to SDS-PAGE gel electrophoresis and transferred to PVDF membranes (Bio-Rad Laboratories, Hemel Hempstead, UK) using semi-dry blotting. Membranes were blocked and incubated with primary anti-H3 antibody, o/n at 4 °C, (sc-8654-R, Santa Cruz Biotechnology, Heidelberg, Germany), followed by a secondary biotin-conjugated IgG for 30 min at RT (ab97083, Abcam, Cambridge, UK) and a streptavidin–biotin/alkaline phosphatase complex (Vectastain ABC-Alkaline Phosphatase for 30 min at RT, Vector Laboratories, Burlingame, USA). Histone H3 bands were detected by luminescent ECL substrate (Advansta, San Jose, USA). Resulting band densities were quantified by ImageQuant TL software (GE Healtcare, Little Chalfont, UK), as compared to known concentrations of purified calf thymus H3 (Roche, Basel, Switzerland). This analysis is independent of frequently observed cross reactivity of histone antibodies with non-histone plasma proteins and allows the inspection of potential in vivo histone proteolytic processing.

The presence of histones was determined via a semi-quantitative Western blotting method as previously described [32 (link),33 (link)]. In short, plasma was diluted 10 times and separated via SDS-PAGE gel electrophoresis (4–15% gradient gel), and transferred to a PVDF membrane (Bio-Rad Laboratories, Hemel Hempstead, UK) using semi-dry blotting. After blocking, the membranes were incubated overnight with a primary rabbit anti-histone H3 antibody (1:10,000, sc-8654-R, Santa Cruz Biotechnology, Heidelberg, Germany), followed by 1 h incubation with a secondary biotin-conjugated donkey anti-rabbit IgG antibody (1:10,000, ab97083, Abcam, Cambridge, UK), and 30 min with a streptavidin-biotin complex (1:500, Vectastain, Vector Laboratories, Burlingame, CA, USA). Histone H3 bands were visualised by the WesternBright ECL substrate (Advansta, San Jose, CA, USA) on the iBright CL1500 Imaging System (ThermoFisher Scientific, Waltham, MA, USA). The band densities were quantified by iBright Analysis Software, compared to known standard concentrations of purified calf thymus H3 (Roche, Basel, Switzerland).