high binding elisa microplates greiner bio-one  (Greiner Bio)

Journal: Basic Research in Cardiology

Article Title: Alkaline nucleoplasm facilitates contractile gene expression in the mammalian heart

doi: 10.1007/s00395-022-00924-9

Figure Lengend Snippet: Transcriptomics identify a subset of pH-responsive cardiac genes and pathways. A Protocol for determining the pH-responsive transcriptome in neonatal ventricular myocytes (NRVMs). Lysates were collected after 48 h of culture in serum-free medium at one of five pH levels. B Analysis of RNAseq performed on total RNA isolated from NRVMs. Histogram of transcripts by expression level, stratified into four groups according to abundance, demonstrating no overall trend in gene expression. Heatmap of differentially expressed genes (DEGs) analysed by DESeq2 using a model with multiple levels of a condition (pH). C Volcano plot of pH-responsive DEGs. Genes in blue are a selection of DEGs with a high combination of significance and fold-change, and abundance greater than the median of all DEGs. Fold-change relates to the ratio of signal at acidic and alkaline pH. D Gene enrichment analysis identified gene ontology (GO) biological functions associated with pH-responsiveness. GO:0006941: striated muscle contraction; GO:1903312: negative regulation of mRNA metabolic process; GO:1903076: regulation of protein localization to plasma membrane; GO:0010970: transport along microtubule; GO:0022607: cellular component assembly; GO:0017158: regulation of calcium ion-dependent exocytosis; GO:0007127: meiosis I; GO:0042982: amyloid precursor protein metabolic process; GO:0086001: cardiac muscle cell action potential; GO:0044839: cell cycle G2/M phase transition. E Volcano plot of pH-responsive DEGs indicating genes of the “striated muscle contraction” ontology (magenta) and genes selected for verification by ELISA inb subsequent experiments (cyan)

Article Snippet: Equal amounts (typically 30 μg) of total protein/well were pipetted into 96-well high-binding ELISA microplate (Greiner Bio-One) and topped up to 50 μl with PBS and left to bind overnight at 37 °C.

Techniques: Isolation, Expressing, Gene Expression, Selection, Clinical Proteomics, Membrane, Sublimation, Enzyme-linked Immunosorbent Assay

Journal: Basic Research in Cardiology

Article Title: Alkaline nucleoplasm facilitates contractile gene expression in the mammalian heart

doi: 10.1007/s00395-022-00924-9

Figure Lengend Snippet: Validating the pH-sensitivity of troponin and Crip2 genes. A Western blot of whole-cell lysates collected from NRVMs after 48 h of culture in serum-free medium at one of four test pH levels for cardiac troponin-T (cTnT; Tnnt2 ), cardiac troponin-I (cTnI; Tnni3 ) and slow skeletal troponin-I (ssTnI; Tnni1 ). β-actin was re-developed using the same membrane as that used for ssTnI. B ELISA absorbance for cTnT, cTnI and ssTnI, and β-actin as a function of pH, normalized to mean signal (average from 4 isolations). ** P < 0.01 and * P < 0.05 by ANOVA. C CRIP2 protein quantified by whole-cell ELISA, showing similar pH-dependence to transcript level (4 repeats). D Western blot of whole-cell lysates collected from NRVMs after 48 h of culture in serum-free medium at either pH 6.4 or 7.44. Each pair represents an independent isolation (i.e. biological repeat). Fractionated lysates showing pH-sensitivity of CRIP2 in the nucleus and cytoplasm, using lamin A/C and GAPDH as loading controls. E CRIP2 western blot of nuclear fractions of NRVM lysates confirm robust pH-responsiveness. F Immunofluorescence imaging of NRVM monolayers for CRIP2, G ssTnI (a pH-responsive DEG/DAP) and H G6PDH (a pH-insensitive protein). Red outlines indicate nuclear regions (Hoechst-33342). (I) Blot for NRVM lysates prepared after immunoprecipitation with CRIP2 antibody, following incubation at pH 6.4 or 7.4. IP blot compared to input. J Silver-stained gel produced from CRIP2 immunoprecipitation, highlighting gel areas selected for mass spectrometry. K Results of mass spectrometry analysis, highlighting proteins involved in contraction. Only proteins that were absent in the negative control (without CRIP2 antibody) but present in the IP are listed

Article Snippet: Equal amounts (typically 30 μg) of total protein/well were pipetted into 96-well high-binding ELISA microplate (Greiner Bio-One) and topped up to 50 μl with PBS and left to bind overnight at 37 °C.

Techniques: Western Blot, Membrane, Enzyme-linked Immunosorbent Assay, Isolation, Immunofluorescence, Imaging, Immunoprecipitation, Incubation, Staining, Produced, Mass Spectrometry, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Exposure to 2.45 GHz Radiation Triggers Changes in HSP-70, Glucocorticoid Receptors and GFAP Biomarkers in Rat Brain

doi: 10.3390/ijms22105103

Figure Lengend Snippet: The histograms represent HSP-70 levels detected by ELISA in the somatosensory cortex ( A ) and the limbic cortex ( B ), 1.5 h (Group A) and 24 h (Group B) after acute exposure and 1.5 h after final repeated exposure (Group C) to a 2.45 GHz signal at 0 and 3 W. Each bar represents mean ± SEM ( n = 24 samples/per group). Asterisks (*) indicate statistically significant differences ( p < 0.05) between the irradiated and control for each group. a, b, c indicate statistically significant differences ( p < 0.05) between irradiated or nonirradiated animals comparing between the different groups A, B, C, which were determined using two-way ANOVA followed by the Holm-Sidak test for multiple comparisons The photographs show the HSP-70 immunomarkers for groups A, B and C in the somatosensory cortex, control (A.1, A.3 and A.5) and irradiated animals (A.2, A.4 and A.6); in the limbic cortex, control (B.1, B.3, B.5) and irradiated animals (B.2, B.4, B.6) ( n = 15 samples/per group). In the photographs it is indicate with the arrowhead and asterisk that means increase on regional cytolocalization. Scale bar = 60 µm.

Article Snippet: One-microgram (μg) aliquots of protein extract in 100 μL of carbonate-bicarbonate buffer (pH 9.6) were placed in the 96 wells of standard ELISA microplates (Greiner Bio-One High-Binding) and incubated overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Irradiation, Control

Journal: International Journal of Molecular Sciences

Article Title: Exposure to 2.45 GHz Radiation Triggers Changes in HSP-70, Glucocorticoid Receptors and GFAP Biomarkers in Rat Brain

doi: 10.3390/ijms22105103

Figure Lengend Snippet: The histograms represent HSP-70 levels detected by ELISA in the hypothalamus ( C ) and the hippocampus ( D ) 1.5 h (Group A) and 24 h (Group B) after acute exposure and 1.5 h after final repeated exposure (Group C) to a 2.45 GHz signal at 0 and 3 W. Each bar represents mean ± SEM ( n = 24 samples/per group). Asterisks (*) indicate statistically significant differences ( p < 0.05) between the irradiated and control for each group. a, b, c, d indicate statistically significant differences ( p < 0.05) between irradiated or nonirradiated animals comparing between the different groups A, B, C, which were determined using two-way ANOVA followed by the Holm-Sidak test for multiple comparisons. The photographs show the HSP-70 immunomarkers for groups A, B and C in the hypothalamus, control (C.1, C.3 and C.5) and irradiated animals (C.2, C.4 and C.6); in the hippocampus, control (D.1, D.3 and D.5) and irradiated animals (D.2, D.4 and D.6) ( n = 15 samples/per group). In the photos it is indicate with the arrowhead and asterisk that means increase on regional cytolocalization. Scale bar = 60 µm.

Article Snippet: One-microgram (μg) aliquots of protein extract in 100 μL of carbonate-bicarbonate buffer (pH 9.6) were placed in the 96 wells of standard ELISA microplates (Greiner Bio-One High-Binding) and incubated overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Irradiation, Control

Journal: International Journal of Molecular Sciences

Article Title: Exposure to 2.45 GHz Radiation Triggers Changes in HSP-70, Glucocorticoid Receptors and GFAP Biomarkers in Rat Brain

doi: 10.3390/ijms22105103

Figure Lengend Snippet: The histograms represent glucocorticoid receptor (GCR) levels detected by ELISA in the somatosensory cortex ( A ) and the limbic cortex ( B ), 1.5 h (Group A) and 24 h (Group B) after acute exposure and 1.5 h after repeated exposure (Group C) to a 2.45 GHz signal at 0 and 3 W. Each bar represents mean ± SEM ( n = 24 samples/per group). Asterisks (*) indicate statistically significant differences ( p < 0.05) between the irradiated and control for each group. a, b, c, d indicate statistically significant differences ( p < 0.05) between irradiated or nonirradiated animals comparing between the different groups A, B, C, which were determined using two-way ANOVA followed by the Holm-Sidak test for multiple comparisons. The photographs show the GCR immunomarkers for groups A, B and C in the somatosensory cortex, control (A.1, A.3 and A.5) and irradiated animals (A.2, A.4 and A.6); in the limbic cortex, control (B.1, B.3 and B.5) and irradiated animals (B.2, B.4 and B.6) ( n = 15 samples/per group). In the photos it is indicate with the arrowhead and asterisk that means increase on regional cytolocalization. Scale bar = 60 µm.

Article Snippet: One-microgram (μg) aliquots of protein extract in 100 μL of carbonate-bicarbonate buffer (pH 9.6) were placed in the 96 wells of standard ELISA microplates (Greiner Bio-One High-Binding) and incubated overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Irradiation, Control

Journal: International Journal of Molecular Sciences

Article Title: Exposure to 2.45 GHz Radiation Triggers Changes in HSP-70, Glucocorticoid Receptors and GFAP Biomarkers in Rat Brain

doi: 10.3390/ijms22105103

Figure Lengend Snippet: The histograms represent glucocorticoid receptor (GCR) levels detected by ELISA in the hypothalamus ( C ) and the hippocampus ( D ) at 1.5 h (Group A) and 24 h (Group B) after acute exposure and 1.5 h after final repeated exposure (Group C) to a 2.45 GHz signal at 0 and 3 W. Each bar represents mean ± SEM ( n = 24 samples/per group). Asterisks (*) indicate statistically significant differences ( p < 0.05) between the irradiated and control for each group. a, b, c indicate statistically significant differences ( p < 0.05) between irradiated or nonirradiated animals comparing between the different groups A, B, C, which were determined using two-way ANOVA followed by the Holm-Sidak test for multiple comparisons. The photographs show the GCR immunomarkers for group A, B and C in the hypothalamus, control (C.1, C.3 and C.5) and irradiated animals (C.2, C.4 and C.6) in the hippocampus, control (D.1, D.3 and D.5) and irradiated animals (D.2, D.4 and D.6) ( n = 15 samples/per group). In the photos it is indicate with the arrowhead and asterisk that means increase on regional cytolocalization. Scale bar = 60 µm.

Article Snippet: One-microgram (μg) aliquots of protein extract in 100 μL of carbonate-bicarbonate buffer (pH 9.6) were placed in the 96 wells of standard ELISA microplates (Greiner Bio-One High-Binding) and incubated overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Irradiation, Control

Journal: International Journal of Molecular Sciences

Article Title: Exposure to 2.45 GHz Radiation Triggers Changes in HSP-70, Glucocorticoid Receptors and GFAP Biomarkers in Rat Brain

doi: 10.3390/ijms22105103

Figure Lengend Snippet: Glial fibrillary acidic protein (GFAP) levels detected by ELISA in the somatosensory cortex ( A ), the limbic cortex ( B ), 1.5 h (Group A) and 24 h (Group B) after acute exposure and 1.5 h after final repeated exposure (Group C) to a 2.45 GHz signal at 0 and 3 W. Each bar represents mean ± SEM. ( n = 24 samples/per group) Asterisks (*) indicate statistically significant differences ( p < 0.05) between the irradiated and control for each group. a, b, c, d indicate statistically significant differences ( p < 0.05) between irradiated or nonirradiated animals comparing between the different groups A, B, C, which were determined using two-way ANOVA followed by the Holm-Sidak test for multiple comparisons. The photographs show the GFAP immunomarkers for groups A, B and C in the somatosensory cortex, control (A.1, A.3 and A.5) and irradiated animals (A.2, A.4 and A.6); in the limbic cortex, control (B.1, B.3 and B.5) and irradiated animals (B.2, B.4 and B.6) ( n = 15 samples/per group). In the photos it is indicate with the arrowhead and asterisk that means increase on regional cytolocalization. Scale bar = 60 µm.

Article Snippet: One-microgram (μg) aliquots of protein extract in 100 μL of carbonate-bicarbonate buffer (pH 9.6) were placed in the 96 wells of standard ELISA microplates (Greiner Bio-One High-Binding) and incubated overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Irradiation, Control

Journal: International Journal of Molecular Sciences

Article Title: Exposure to 2.45 GHz Radiation Triggers Changes in HSP-70, Glucocorticoid Receptors and GFAP Biomarkers in Rat Brain

doi: 10.3390/ijms22105103

Figure Lengend Snippet: Glial fibrillary acidic protein (GFAP) levels detected by ELISA in the hypothalamus ( C ) and the hippocampus ( D ) 1.5 h (Group A) and 24 h (Group B) after acute exposure and 1.5 h after final repeated exposure (Group C) to a 2.45 GHz signal at 0 and 3 W. Each bar represents mean ± SEM ( n = 24 samples/per group). Asterisks (*) indicate statistically significant differences ( p < 0.05) between the irradiated and control for each group. a, b, c, d indicate statistically significant differences ( p < 0.05) between irradiated or nonirradiated animals comparing between the different groups A, B, C, which were determined using two-way ANOVA followed by the Holm-Sidak test for multiple comparisons. The photographs show the GFAP immunomarkers for groups A, B and C in hypothalamus, control (C.1, C.3 and C.5) and irradiated animals (C.2, C.4 and C.6); in hippocampus, control (D.1, D.3 and D.5) and irradiated animals (D.2, D.4 and D.6) ( n = 15 samples/per group). In the photos it is indicate with the arrowhead and asterisk that means increase on regional cytolocalization. Scale bar = 60 µm.

Article Snippet: One-microgram (μg) aliquots of protein extract in 100 μL of carbonate-bicarbonate buffer (pH 9.6) were placed in the 96 wells of standard ELISA microplates (Greiner Bio-One High-Binding) and incubated overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Irradiation, Control

Journal: International Journal of Molecular Sciences

Article Title: Exposure to 2.45 GHz Radiation Triggers Changes in HSP-70, Glucocorticoid Receptors and GFAP Biomarkers in Rat Brain

doi: 10.3390/ijms22105103

Figure Lengend Snippet: Experimental design. HSP-70: Heat shock protein-70, GCR: glucocorticoid receptor, GFAP: Glial fibrillary acidic protein, CLIA: Chemiluminescent Enzyme-Linked Immunosorbent Assay, Immunohistochemistry.

Article Snippet: One-microgram (μg) aliquots of protein extract in 100 μL of carbonate-bicarbonate buffer (pH 9.6) were placed in the 96 wells of standard ELISA microplates (Greiner Bio-One High-Binding) and incubated overnight at 4 °C.

Techniques: Chemiluminescent ELISA, Immunohistochemistry