Highly reliable quantification of proteins such as members of the HSP70 superfamily based on the grey scale index via immune detection stained bands on a Western blot
Introduction
The heat-shock-protein 70 (HSP70) superfamily is known as a component of the innate immune system [1], [2]. HSC70, a constitutive stress protein and HSP70, an induced stress protein, are the most common proteins of this superfamily [3]. As part of the immune system these proteins are a cellular response to stress factors from the environment (e.g. hyperthermia, hypothermia, hypoxia, ischaemia) [4]. To prevent damages to the cells protein biosynthesis the stress proteins bind to defected proteins and rearrange their tertiary structure into their genuine form [5], [6]. Because of the immediate reaction to stress factors the HSP70 superfamily is usable as a biomarker for hyperthermia, hypothermia and hypoxia [7], [8], [9]. In previous investigations Enzyme-linked Immunosorbent Assays (ELISA) and immunohistochemically staining methods were used to locate and quantify protein concentrations [10], [11].
As the costs for reagents and devices as well as the work schedule of these methods are immense it was the goal of our study to develop an easy and reliable method to quantify the concentration of specific proteins. We established a procedure to measure the relative concentration of proteins fixed on ROTI® PVDF (ROTH™) membranes. This new method was then applied to check for degradation of specific HSP70 proteins due to storage and freezing processes.
Section snippets
Materials and methods
From 56 individuals (average life span 57 years, both genders) samples from cerebrum and cerebellum (each about 100 g) were taken during autopsies. Only cases were chosen in which tissues were used for routine analysis. Whole proteins were extracted from 100 mg cerebrum and cerebellum tissue according to Ikeda et al. 1998. An amount of 100 μg total proteins per sample were then loaded on 10% polyacrylamide/30% bisacrylamide gels and separated at 100 V, 140 mA and 80 W for 120 min [12], [13], [14].
The
Results and discussion
Using the above calculated conversion factor and the Membralyzer© software it is possible to measure the relative concentration of a specific protein in a sample with defined total protein concentration. The grey scale indices used for the measurement with the Membralyzer© contain a range of 0 up to 255 GSI. Thus we are able to reach a sensitivity of 0.18 μg/μl down to 0.11 μg/μl for a quantification using our parameters. The measurements were taken in triplicates for each band. Every marked band
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Cited by (2)
Detection of constitutive and inducible HSP70 proteins in formalin fixed human brain tissue
2014, Forensic Science InternationalCitation Excerpt :To test the sensitivity of this assay, a serial dilution (protein concentrations from 200 μg/μl down to 5 μg/μl) was analyzed by Western blotting for the primary antibody (anti-HSPA1A–HSPA8). Specific HSP70 proteins were then quantified using the self-developed software Membralyzer© version 2.5.1 as recently published [19]. For immmunohistochemical investigations all formalin-fixed tissues were dehydrated (70% ethanol for 1 h; 70% ethanol for 2 h; 96% ethanol two times for 2 h; three times absolute alcohol for 1.5 h, 2 h and 2.5 h; three times Xylol for 1.5 h, 2 h and 2.5 h and finally two times paraffin for 2 h each) in a Shandon, Hypercenter XP (Thermo Scientific, Germany) overnight.