Journal: PLoS Genetics

Article Title: Duplicate divergence of two bacterial small heat shock proteins reduces the demand for Hsp70 in refolding of substrates

doi: 10.1371/journal.pgen.1008479

Figure Lengend Snippet: (A) The distribution of sHsp genes ( ibpA red; ibpB blue) at the species phylogeny of γ-proteobacteria (calculated with a supermatrix of 1500 orthologs from 50 γ-proteobacteria genomes using Maximum Likelihood method with GTR+GAMMA model) indicates the presence of a single copy ibpA (black) in γ-proteobacteria before the speciation of Enterobacterales , both ibpA and ibpB (green) are co-expressed in the subset of Enterobacterales ( Hafniaceae , Pectobacteriaceae , Yersiniaceae , Enterobacteriaceae ), and ibpB is absent in Erwiniaceae (purple). Based on parsimony two evolutionary events in γ-proteobacteria were inferred, the duplication of ibpA at the base of Enterobacterales (star) and loss of ibpB in Erwiniacea (cross). (B) The evolutionary history of sHsps (IbpA red; IbpB blue) in γ-proteobacteria, calculated with Maximum Likelihood method with CAT+I+LG model, indicates the presence single duplication event at the base of Enterobacterales , resulting in a presence of both IbpA and IbpB in Hafniaceae , Pectobacteriaceae , Yersiniaceae , Enterobacteriaceae (green). In all these lineages ibpB evolves faster than ibpA , but no ibpB is present in Erwiniacea (purple). Scale bar indicates number of substitution per site.

Article Snippet: C . neteri ibpA and ibpB together with A . hydrophila ibpA genes were ordered from Genscript (cloned into pET3a plasmids).

Techniques:

Journal: PLoS Genetics

Article Title: Duplicate divergence of two bacterial small heat shock proteins reduces the demand for Hsp70 in refolding of substrates

doi: 10.1371/journal.pgen.1008479

Figure Lengend Snippet: (A) DLS measurements of sHsps-luciferase assemblies. Average hydrodynamic radius (±SD) of the most occupied peak (min. 80% of total volume) from DLS size distributions of sHsps-luciferase assemblies. sHsps-luciferase assemblies were prepared as follows: luciferase at fixed 1.5 μM concentration in the presence of sHsps (0 to 10 μM as depicted) was denatured at 44°C for 10 min. When IbpA Ec and IbpB Ec were tested together (IbpAB Ec ), the 1:2 stoichiometry was used (e.g. 10 μM IbpAB Ec is 3.33 μM IbpA Ec and 6.67 μM IbpB Ec ). (B) Sedimentation profiles of sHsps-luciferase assemblies. 1 μM luciferase was denatured at 44°C for 10 min in the presence of 5 μM sHsps and subjected to sedimentation in glycerol gradient. Fractions were collected from the top and analysed by SDS-PAGE followed by Oriole staining.

Article Snippet: C . neteri ibpA and ibpB together with A . hydrophila ibpA genes were ordered from Genscript (cloned into pET3a plasmids).

Techniques: Luciferase, Concentration Assay, Sedimentation, SDS Page, Staining

Journal: PLoS Genetics

Article Title: Duplicate divergence of two bacterial small heat shock proteins reduces the demand for Hsp70 in refolding of substrates

doi: 10.1371/journal.pgen.1008479

Figure Lengend Snippet: (A) Experimental scheme. (B) Aggregated luciferase immobilized on the BLI sensor was associated with sHsps as in and subsequently introduced to buffer containing limiting (DnaK 0.7 μM; DnaJ 0.28 μM; GrpE 0.21 μM) (upper panels) or saturating (DnaK 3.5 μM; DnaJ 1.4 μM; GrpE 1.05 μM) (lower panels) Hsp70 machinery concentrations (dark traces). Light traces represent spontaneous sHsps dissociation curves (as shown in ), grey dashed traces show Hsp70 machinery binding to immobilized luciferase aggregates in the absence of any sHsps. (C) Sensor-bound proteins were analysed before and after Hsp70 machinery action with SDS-PAGE followed by Western blot using IbpA or IbpB antibodies and Oriole staining for DnaK and luciferase. No incubation lanes refer to proteins associated with the sensor prior to incubation with either limiting or saturating Hsp70 concentration. The experiments presented in panel B were repeated at least twice, supporting SDS-PAGE (Oriole stained) and Western blot analyses (panel C) were performed once.

Article Snippet: C . neteri ibpA and ibpB together with A . hydrophila ibpA genes were ordered from Genscript (cloned into pET3a plasmids).

Techniques: Luciferase, Binding Assay, SDS Page, Western Blot, Staining, Incubation, Concentration Assay

Journal: PLoS Genetics

Article Title: Duplicate divergence of two bacterial small heat shock proteins reduces the demand for Hsp70 in refolding of substrates

doi: 10.1371/journal.pgen.1008479

Figure Lengend Snippet: (A) Expression of IbpA and IbpB in constructed strains. E . coli MC4100 WT, Δ ibpA , Δ ibpB and Δ ibpAB cultures grown in 30°C were subjected to heat shock (42°C, 10 min and 48°C, 5 min) and analysed with SDS-PAGE followed by western blot against IbpA and IbpB. (B) Localization of IbpA and IbpB in soluble and aggregated protein fractions. WT, Δ ibpA and Δ ibpB strains were heat-shocked as in A, followed by isolation of soluble and aggregated protein fractions as described in Methods. Obtained fractions were then analysed by SDS-PAGE and Western blot. (C) IbpB presence allows for IbpA removal from aggregates. Bacterial strains were heat-shocked as in A and allowed to recover at 30°C. Aggregated and soluble protein fractions were isolated from culture aliquots sampled at indicated time points and further analysed by SDS-PAGE Coomassie staining and Western blot using antibodies against IbpA or IbpB. Quantifications of total aggregated proteins (Aggr. prot.) and sHsps (both Western blot -Wb and Coomassie blue -Cb analysis) were plotted against recovery time.

Article Snippet: C . neteri ibpA and ibpB together with A . hydrophila ibpA genes were ordered from Genscript (cloned into pET3a plasmids).

Techniques: Expressing, Construct, SDS Page, Western Blot, Isolation, Staining

Journal: Proteomes

Article Title: The New and the Old: Platform Cross-Validation of Immunoaffinity MASS Spectrometry versus ELISA for PromarkerD, a Predictive Test for Diabetic Kidney Disease

doi: 10.3390/proteomes8040031

Figure Lengend Snippet: IAMS and ELISA working ranges and dilutions.

Article Snippet: The ELISA assay for the detection of APOA4, CD5L and IBP3 was developed by TGR BioSciences (Adelaide, SA, Australia).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Proteomes

Article Title: The New and the Old: Platform Cross-Validation of Immunoaffinity MASS Spectrometry versus ELISA for PromarkerD, a Predictive Test for Diabetic Kidney Disease

doi: 10.3390/proteomes8040031

Figure Lengend Snippet: Difference plot ( A ) before Bland Altman correction comparing IBP3 concentrations derived from IAMS and ELISA platforms. Scatter plot ( B ) after adjusting ELISA concentration values with a Bland Altman analysis. (adj = adjustment of division by 9.52 µg/mL to the ELISA value).

Article Snippet: The ELISA assay for the detection of APOA4, CD5L and IBP3 was developed by TGR BioSciences (Adelaide, SA, Australia).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Clinical Proteomics

Article Title: A robust multiplex immunoaffinity mass spectrometry assay (PromarkerD) for clinical prediction of diabetic kidney disease

doi: 10.1186/s12014-020-09302-w

Figure Lengend Snippet: Immunoaffinity assay temperature, freeze-thaw, and extract, stability

Article Snippet: Monoclonal antibody targeting the biomarker APOA4 was developed by the Monash Antibody Technologies Facility (MATF, Monash University, Melbourne, Australia) and monoclonal antibodies targeting CD5L and IBP3 were developed by CDI laboratories (Mayaguez, Puerto Rico).

Techniques:

Journal: Clinical Proteomics

Article Title: A robust multiplex immunoaffinity mass spectrometry assay (PromarkerD) for clinical prediction of diabetic kidney disease

doi: 10.1186/s12014-020-09302-w

Figure Lengend Snippet: Difference plots for original immunodepletion (ImDe) method and new immunoaffinity (IAMS) method biomarker concentrations. Panel A is for APOA4, panel B for CD5L and panel C for IBP3. These difference plots show the agreement between methods for each biomarker, after IAMS concentrations were adjusted based on the mean bias determined from Bland Altman plot analysis. LoA is Limits of Agreement. 95%CI is the 95% Confidence Interval

Article Snippet: Monoclonal antibody targeting the biomarker APOA4 was developed by the Monash Antibody Technologies Facility (MATF, Monash University, Melbourne, Australia) and monoclonal antibodies targeting CD5L and IBP3 were developed by CDI laboratories (Mayaguez, Puerto Rico).

Techniques: Biomarker Assay