To generate samples for proteomic analysis, the frozen biological samples were pulverized in a mortar containing liquid nitrogen to prevent thawing. The resulting powder was solubilised in 330 μl lysis buffer (2 mol/l Thiourea, 6 mol/l Urea, 4% CHAPS, 1 cOmplete ULTRA Tablets Mini (Roche, Penzberg, Germany) per 5 ml buffer). Afterwards, each sample was centrifuged using a QIA Shredder Mini Spin Column (Qiagen, Hilden, Germany) for 3 min at 16,100 r.c.f. to get rid of debris. Proteins were precipitated using 30% trichloroacetic acid for 20 min on ice to inhibit proteolytic activity.22 Subsequently, samples were centrifuged for 10 min at 16,100 r.c.f., the supernatant was discarded and the protein pellet was washed three times with cold acetone. The pellet was dried and resolved in lysis buffer. The pH of the solution was adjusted to 8 by adding 50 mM NaOH. Protein concentration was determined by Bradford Assay (Coomassie Plus (Bradford) Assay Reagent, Thermo Scientific, Braunschweig, Germany) according to the manufacturer’s instructions. To reach sufficient protein concentrations for 2D-difference gel electrophoresis (DIGE), two clinorotation runs per group were pooled leading to five biological replicates.
Coomassie plus bradford assay reagent
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Coomassie Plus Bradford assay reagent is a colorimetric reagent solution used for the quantitative determination of protein concentration. It is based on the Bradford method, which measures the change in absorbance of the Coomassie Brilliant Blue G-250 dye in response to different protein concentrations.
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Lab products found in correlation
Qiashredder mini spin column, by Qiagen (1 mentions)
Image studio 4, by LI COR (1 mentions)
Ecl western blotting substrate kit, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Bio-Rad (1 mentions)
Gel purified pcr products, by Qiagen (1 mentions)
Amicon ultra 15 centrifugal filter, by Merck Group (1 mentions)
Bead mill tube, by Thermo Fisher Scientific (1 mentions)
Protein phosphatase inhibitor, by Abcam (1 mentions)
One shot model, by Constant Systems (1 mentions)
7 protocols using coomassie plus bradford assay reagent
1
Protein Sample Preparation for Proteomic Analysis
2
Western Blot Analysis of Liver Proteins
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For western blot analysis five animals were chosen from each group in random and total protein extracted from the livers and concentrations measured by Coomassie Plus Bradford assay reagent (Thermo Scientific, USA). β-mercaptoethanol was added to a final concentration of 5%, after which each sample was denatured by heating for 10 min (95°C). Next, 40 μg of protein from each sample was added to a 10% sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel. Electrophoresis productions were transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-rad, USA), blocked with 5% bovine serum albumin/0.1% Tween-20, incubated with primary antibody (1:1000) (Sigma, UK) and HRP-conjugated secondary antibody (1:5000) (AbD Serotec, UK), respectively. The proteins were visualized using the ECL Western Blotting substrate kit (Abcam, USA) according to its manufacturer’s instructions. Finally, immunoblotting signals were quantitated using Image Studio 4.0 (LI-COR Biotechnology, USA).
3
Expression and Purification of Francisella tularensis Proteins
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The genes encoding selected F. tularensis proteins were amplified by PCR with specific primers (Supplementary Table S2 ), and the gel-purified PCR products (Qiagen) were inserted into pET28b using either the NcoI and XhoI restriction sites (pepSY, rho, pepB, gene of YCII-related domain protein) or HindIII and XhoI restriction sites (trxA). The resulting plasmids encoded the aforementioned proteins with a C-terminal histidine tag. The final plasmid constructs were verified by direct DNA sequencing. For protein expression, the plasmids were transformed into E. coli NiCo21 expression strains (New England BioLabs, Ipswich, AM, United States). The cells were grown in Terrific Broth medium at 28°C overnight. Bacteria were lysed in a French pressure cell and the lysates were applied to a TALON® Metal Affinity Resin purification system (Clontech, Mountain View, CA, United States) for purification of His-tagged proteins. Finally, the elution buffer was changed to T100N150 (100 mM Tris-HCl, 150 mM NaCl, [pH 7.6]) using an Amicon® Ultra-15 centrifugal filter (Sigma–Aldrich). Eluted proteins were verified by SDS-PAGE followed by Coomassie staining (data not shown), and the protein concentrations were determined using Coomassie Plus Bradford assay reagent (Thermo Fisher).
4
Protein Extraction from Tissue Samples
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A total of 15 MT were added to RIPA lysis buffer supplemented with a complete protease inhibitor mixture and a protein phosphatase inhibitor (Abcam, UK). For tissue homogenisation, pre-filled bead mill tubes (Thermo Fisher, UK) and an evolution homogeniser was used (Percellys, France). The samples were thoroughly mixed and stored on ice for 10 min before centrifugation at 10,000 g for 10 min. The supernatants were transferred to a fresh tube and centrifuged for further 10 min period. The protein concentrations were measured utilising a Coomassie Plus Bradford assay reagent (Thermo Scientific, UK). The supernatants were stored at − 80 °C.
5
Bacterial Cell Lysis and Protein Extraction
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Bacteria were grown in sBHI at 37°C with shaking at 180 rpm. When bacterial cells were in mid-exponential phase, they were harvested by centrifugation at 6,000xg for 15 minutes then resuspended in 10 mM HEPES, pH 7.4 and lysed by one passage through a high-pressure cell (25,000 psi; One Shot Model, Constant Systems Ltd). Insoluble material was removed by centrifugation at 16,000xg for 20 minutes, the clarified supernatant collected and protein concentration assessed using Coomassie Plus (Bradford) Assay Reagent (Thermo Fisher Scientific).
6
Venom Extraction from Spider Females
7
Liver Tissue Homogenization and Protein Extraction
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The liver samples were thawed on ice and homogenized in a homogenization buffer (RIPA lysis buffer supplemented with a complete protease inhibitor mixture and a protein phosphatase inhibitor) (Abcam, UK). For tissue homogenization, prefilled bead mill tubes (Thermo Fisher, UK) and an evolution homogenizer was used (Percellys, France). The samples were thoroughly mixed and stored on ice for 10 min before centrifugation at 10000 g for 10 min. The supernatants were transferred to a fresh tube and centrifuged for a further 10 min. The protein concentrations were measured utilizing a Coomassie Plus Bradford assay reagent (Thermo Scientific, UK) and the supernatants stored at À80 C until use.
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