Structured Review

Bio-Rad fluor s multiimager
Direct demonstration of TBP exchange. (A) Concentration dependence of TBP exchange within TFIID. Multiple, equal amounts of HA-TAF130p-tagged TFIID bound to protein A-Sepharose beads coated with anti-HA monoclonal antibody were generated as detailed in Materials and Methods. These aliquots of bead-bound TFIID were incubated with the indicated increasing molar excess of His 6 -tagged TBP for 30 min. Unbound His 6 -tagged TBP was removed by centrifugation and washing. The relative amounts of wild-type (WT) and His 6 -TBP remaining bead bound to TFIID were determined by SDS-PAGE and immunoblotting with polyclonal anti-TBP IgG. This immunoblot is shown in the inset and labeled WT TBP (TFIID-endogenous TBP) and His 6 -TBP (TBP exchanged into TFIID). Appropriate regions of the gel blot were separately probed with TFIID-specific antibodies (anti-TAF130p, anti-TAF65p, or anti-TAF48p IgGs). All immune complexes were detected by chemiluminescence and quantitated with a <t>Fluor-S</t> Bio-Rad <t>MultiImager.</t> Wild-type and His 6 -TBP content, corrected for average TAF recovery, is plotted as percent maximal signal as a function of His 6 -TBP/wild-type TBP ratio. Background subtracted and recovery-corrected wild-type TBP and His 6 -TBP values, in arbitrary units, were 123 and 0, 126 and 34, 116 and 76, 124 and 136, 92 and 126, 43 and 150, 27 and 161, and 21 and 149 in the reactions with His 6 -TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Average TAFp recoveries in these reactions ranged from 95 to 112%. (B) Kinetics of TBP-TFIID exchange. TBP-TFIID exchange was performed as above. Seven identical reactions were set up, each with a 10-fold molar excess of His 6 -tagged TBP relative to TFIID-endogenous wild-type TBP. Samples were incubated for 0, 10, 20, 30, 45, 60, or 120 min, as indicated. Wild-type and His 6 -TBP levels (upper) and TAFp content (lower) were determined by immunoblotting as in panel A. The levels of wild-type and His 6 -tagged TBP in these reactions were 75 and 0, 53 and 89, 51 and 92, 49 and 87, 38 and 96, 27 and 98, and 25 and 94 arbitrary units in the reactions with His 6 -TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Recovery of TAFs (lower) was in the same range as for panel A.
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Images

1) Product Images from "Molecular Characterization of Saccharomyces cerevisiae TFIID"

Article Title: Molecular Characterization of Saccharomyces cerevisiae TFIID

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.16.6000-6013.2002

Direct demonstration of TBP exchange. (A) Concentration dependence of TBP exchange within TFIID. Multiple, equal amounts of HA-TAF130p-tagged TFIID bound to protein A-Sepharose beads coated with anti-HA monoclonal antibody were generated as detailed in Materials and Methods. These aliquots of bead-bound TFIID were incubated with the indicated increasing molar excess of His 6 -tagged TBP for 30 min. Unbound His 6 -tagged TBP was removed by centrifugation and washing. The relative amounts of wild-type (WT) and His 6 -TBP remaining bead bound to TFIID were determined by SDS-PAGE and immunoblotting with polyclonal anti-TBP IgG. This immunoblot is shown in the inset and labeled WT TBP (TFIID-endogenous TBP) and His 6 -TBP (TBP exchanged into TFIID). Appropriate regions of the gel blot were separately probed with TFIID-specific antibodies (anti-TAF130p, anti-TAF65p, or anti-TAF48p IgGs). All immune complexes were detected by chemiluminescence and quantitated with a Fluor-S Bio-Rad MultiImager. Wild-type and His 6 -TBP content, corrected for average TAF recovery, is plotted as percent maximal signal as a function of His 6 -TBP/wild-type TBP ratio. Background subtracted and recovery-corrected wild-type TBP and His 6 -TBP values, in arbitrary units, were 123 and 0, 126 and 34, 116 and 76, 124 and 136, 92 and 126, 43 and 150, 27 and 161, and 21 and 149 in the reactions with His 6 -TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Average TAFp recoveries in these reactions ranged from 95 to 112%. (B) Kinetics of TBP-TFIID exchange. TBP-TFIID exchange was performed as above. Seven identical reactions were set up, each with a 10-fold molar excess of His 6 -tagged TBP relative to TFIID-endogenous wild-type TBP. Samples were incubated for 0, 10, 20, 30, 45, 60, or 120 min, as indicated. Wild-type and His 6 -TBP levels (upper) and TAFp content (lower) were determined by immunoblotting as in panel A. The levels of wild-type and His 6 -tagged TBP in these reactions were 75 and 0, 53 and 89, 51 and 92, 49 and 87, 38 and 96, 27 and 98, and 25 and 94 arbitrary units in the reactions with His 6 -TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Recovery of TAFs (lower) was in the same range as for panel A.
Figure Legend Snippet: Direct demonstration of TBP exchange. (A) Concentration dependence of TBP exchange within TFIID. Multiple, equal amounts of HA-TAF130p-tagged TFIID bound to protein A-Sepharose beads coated with anti-HA monoclonal antibody were generated as detailed in Materials and Methods. These aliquots of bead-bound TFIID were incubated with the indicated increasing molar excess of His 6 -tagged TBP for 30 min. Unbound His 6 -tagged TBP was removed by centrifugation and washing. The relative amounts of wild-type (WT) and His 6 -TBP remaining bead bound to TFIID were determined by SDS-PAGE and immunoblotting with polyclonal anti-TBP IgG. This immunoblot is shown in the inset and labeled WT TBP (TFIID-endogenous TBP) and His 6 -TBP (TBP exchanged into TFIID). Appropriate regions of the gel blot were separately probed with TFIID-specific antibodies (anti-TAF130p, anti-TAF65p, or anti-TAF48p IgGs). All immune complexes were detected by chemiluminescence and quantitated with a Fluor-S Bio-Rad MultiImager. Wild-type and His 6 -TBP content, corrected for average TAF recovery, is plotted as percent maximal signal as a function of His 6 -TBP/wild-type TBP ratio. Background subtracted and recovery-corrected wild-type TBP and His 6 -TBP values, in arbitrary units, were 123 and 0, 126 and 34, 116 and 76, 124 and 136, 92 and 126, 43 and 150, 27 and 161, and 21 and 149 in the reactions with His 6 -TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Average TAFp recoveries in these reactions ranged from 95 to 112%. (B) Kinetics of TBP-TFIID exchange. TBP-TFIID exchange was performed as above. Seven identical reactions were set up, each with a 10-fold molar excess of His 6 -tagged TBP relative to TFIID-endogenous wild-type TBP. Samples were incubated for 0, 10, 20, 30, 45, 60, or 120 min, as indicated. Wild-type and His 6 -TBP levels (upper) and TAFp content (lower) were determined by immunoblotting as in panel A. The levels of wild-type and His 6 -tagged TBP in these reactions were 75 and 0, 53 and 89, 51 and 92, 49 and 87, 38 and 96, 27 and 98, and 25 and 94 arbitrary units in the reactions with His 6 -TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Recovery of TAFs (lower) was in the same range as for panel A.

Techniques Used: Concentration Assay, Generated, Incubation, Centrifugation, SDS Page, Labeling, Western Blot

2) Product Images from "SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1 [W]"

Article Title: SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1 [W]

Journal: Plant Physiology

doi: 10.1104/pp.108.118174

Chlorophyll and anthocyanin contents and the expression of light-inducible genes in shw1 mutants. A, Accumulation of chlorophyll in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings. B, Accumulation of anthocyanin in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings. C, Quantification of RNA gel-blot analysis data of CAB1 in wild type (Col) and shw1-1 mutant seedlings. Five-day-old dark-grown seedlings were transferred to WL for 2, 4, and 8 h. Five-day-old dark-grown seedlings have been shown as 0 h. The experiment was repeated three times. The intensity of each band in autoradiograph was quantified by the Fluor-S-MultiImager (Bio-Rad) and ratios of the CAB1 gene versus its corresponding 18S rRNA band were determined and plotted (Fluor-S-MultiImager; Bio-Rad). D, Quantification of RNA gel-blot analysis data of RBCS in wild type (Col) and shw1-1 mutant seedlings. For experimental detail see legend to Figure 5C. E, Accumulation of chlorophyll in the cotyledons after normalized by cotyledon size in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings.
Figure Legend Snippet: Chlorophyll and anthocyanin contents and the expression of light-inducible genes in shw1 mutants. A, Accumulation of chlorophyll in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings. B, Accumulation of anthocyanin in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings. C, Quantification of RNA gel-blot analysis data of CAB1 in wild type (Col) and shw1-1 mutant seedlings. Five-day-old dark-grown seedlings were transferred to WL for 2, 4, and 8 h. Five-day-old dark-grown seedlings have been shown as 0 h. The experiment was repeated three times. The intensity of each band in autoradiograph was quantified by the Fluor-S-MultiImager (Bio-Rad) and ratios of the CAB1 gene versus its corresponding 18S rRNA band were determined and plotted (Fluor-S-MultiImager; Bio-Rad). D, Quantification of RNA gel-blot analysis data of RBCS in wild type (Col) and shw1-1 mutant seedlings. For experimental detail see legend to Figure 5C. E, Accumulation of chlorophyll in the cotyledons after normalized by cotyledon size in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings.

Techniques Used: Expressing, Mutagenesis, Western Blot, Autoradiography

3) Product Images from "SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1 [W]"

Article Title: SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1 [W]

Journal: Plant Physiology

doi: 10.1104/pp.108.118174

Chlorophyll and anthocyanin contents and the expression of light-inducible genes in shw1 mutants. A, Accumulation of chlorophyll in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings. B, Accumulation of anthocyanin in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings. C, Quantification of RNA gel-blot analysis data of CAB1 in wild type (Col) and shw1-1 mutant seedlings. Five-day-old dark-grown seedlings were transferred to WL for 2, 4, and 8 h. Five-day-old dark-grown seedlings have been shown as 0 h. The experiment was repeated three times. The intensity of each band in autoradiograph was quantified by the Fluor-S-MultiImager (Bio-Rad) and ratios of the CAB1 gene versus its corresponding 18S rRNA band were determined and plotted (Fluor-S-MultiImager; Bio-Rad). D, Quantification of RNA gel-blot analysis data of RBCS in wild type (Col) and shw1-1 mutant seedlings. For experimental detail see legend to Figure 5C. E, Accumulation of chlorophyll in the cotyledons after normalized by cotyledon size in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings.
Figure Legend Snippet: Chlorophyll and anthocyanin contents and the expression of light-inducible genes in shw1 mutants. A, Accumulation of chlorophyll in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings. B, Accumulation of anthocyanin in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings. C, Quantification of RNA gel-blot analysis data of CAB1 in wild type (Col) and shw1-1 mutant seedlings. Five-day-old dark-grown seedlings were transferred to WL for 2, 4, and 8 h. Five-day-old dark-grown seedlings have been shown as 0 h. The experiment was repeated three times. The intensity of each band in autoradiograph was quantified by the Fluor-S-MultiImager (Bio-Rad) and ratios of the CAB1 gene versus its corresponding 18S rRNA band were determined and plotted (Fluor-S-MultiImager; Bio-Rad). D, Quantification of RNA gel-blot analysis data of RBCS in wild type (Col) and shw1-1 mutant seedlings. For experimental detail see legend to Figure 5C. E, Accumulation of chlorophyll in the cotyledons after normalized by cotyledon size in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings.

Techniques Used: Expressing, Mutagenesis, Western Blot, Autoradiography

4) Product Images from "Molecular and Cellular Characterization of the Tomato Pollen Profilin, LePro1"

Article Title: Molecular and Cellular Characterization of the Tomato Pollen Profilin, LePro1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0086505

Immunoblot analysis of total soluble proteins extracted from pollen grains of wildtype (C), antisense (A1–A3) and sense (S1–5) plants. Ten micrograms of proteins per sample were loaded in each well. A mixture of two antibodies, monoclonal anti-actin antibody 3H11 and polyclonal anti-profilin antibody Tp1 were used. A , actin (43 kD) and profilin (14 kD) were detected within the same blot. The upper bands represent actin signals and the lower bands represent profilin signals. The left column shows molecular weight standards (M) in kD. B , protein signal intensities obtained with a Bio-Rad Fluor-S MultiImager densitometer.
Figure Legend Snippet: Immunoblot analysis of total soluble proteins extracted from pollen grains of wildtype (C), antisense (A1–A3) and sense (S1–5) plants. Ten micrograms of proteins per sample were loaded in each well. A mixture of two antibodies, monoclonal anti-actin antibody 3H11 and polyclonal anti-profilin antibody Tp1 were used. A , actin (43 kD) and profilin (14 kD) were detected within the same blot. The upper bands represent actin signals and the lower bands represent profilin signals. The left column shows molecular weight standards (M) in kD. B , protein signal intensities obtained with a Bio-Rad Fluor-S MultiImager densitometer.

Techniques Used: Molecular Weight

5) Product Images from "Glycosphingolipids Promote Entry of a Broad Range of Human Immunodeficiency Virus Type 1 Isolates into Cell Lines Expressing CD4, CXCR4, and/or CCR5"

Article Title: Glycosphingolipids Promote Entry of a Broad Range of Human Immunodeficiency Virus Type 1 Isolates into Cell Lines Expressing CD4, CXCR4, and/or CCR5

Journal: Journal of Virology

doi:

TLC of GSL levels in control and PPMP-treated cells. GSLs were isolated as described in Materials and Methods from GHOST-345 cells that were either untreated or treated for at least 7 days in culture medium containing 10 μM PPMP. Monosialoganglioside standard (10 μg), containing GM3, GM2, and GM1, and lipid extracts from 5 × 10 6 cells were spotted onto a 20- by 20-cm silica gel TLC plate (Fisher, Malvern, Pa.), which was developed in CHCl 3 –CH 3 OH–10% KCl (aq) (50:40:10, vol/vol/vol). After being run, the plate was air dried, sprayed with resorcinol, and heated to develop the spots. Images were taken on a Fluor-S MultiImager using white light epi-illumination. Purple bands, indicative of GSLs interacting with resorcinol, are outlined in the standard and untreated lanes. We have quantified the extent of GSL depletion by determining the integrated grey levels from regions of interest of the Fluor-S MultiImager scans corresponding to the arrows. For the top arrow, the integrated grey levels are 77,806 and 32,295 in lanes 2 and 3, respectively, and for the bottom arrow, they are 120,063 and 38,531 in lanes 2 and 3, respectively.
Figure Legend Snippet: TLC of GSL levels in control and PPMP-treated cells. GSLs were isolated as described in Materials and Methods from GHOST-345 cells that were either untreated or treated for at least 7 days in culture medium containing 10 μM PPMP. Monosialoganglioside standard (10 μg), containing GM3, GM2, and GM1, and lipid extracts from 5 × 10 6 cells were spotted onto a 20- by 20-cm silica gel TLC plate (Fisher, Malvern, Pa.), which was developed in CHCl 3 –CH 3 OH–10% KCl (aq) (50:40:10, vol/vol/vol). After being run, the plate was air dried, sprayed with resorcinol, and heated to develop the spots. Images were taken on a Fluor-S MultiImager using white light epi-illumination. Purple bands, indicative of GSLs interacting with resorcinol, are outlined in the standard and untreated lanes. We have quantified the extent of GSL depletion by determining the integrated grey levels from regions of interest of the Fluor-S MultiImager scans corresponding to the arrows. For the top arrow, the integrated grey levels are 77,806 and 32,295 in lanes 2 and 3, respectively, and for the bottom arrow, they are 120,063 and 38,531 in lanes 2 and 3, respectively.

Techniques Used: Thin Layer Chromatography, Isolation

6) Product Images from "Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation"

Article Title: Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation

Journal: BMC Medical Genomics

doi: 10.1186/1755-8794-2-51

Immunohistochemical stain for HCV core in the 2-month-old double transgenic mouse (DTM) liver with high (A, B), intermediate (C, D) and low (E, F) HCV core protein expression . The upper region of panels G, H and I depict results of Western blots for HCV core protein extracted from the same livers after two months on control chow (permissive diet). Detection in DTM with high (G), intermediate (H), and low HCV core protein expression (I). Lane 1, HeLa cells transfected with HCV core plasmid (positive control, He); lane 2, liver (li); lane 3, heart (h); lane 4, kidney (k); lane 5, thymus (t); lane 6, omental fat (o); lane 7, lung (lu); lane 8, muscle (m); lane 9, intestine (i); lane 10, skin (s). As a loading control, the same samples were probed for GAPDH (lower panel G, H, and I). (J) Quantitative immunoassays for hepatic HCV core protein from high, intermediate, and low protein-expressing mouse lines. Y-axis, core bands densities (units) acquired from Fluor-S multiimager.
Figure Legend Snippet: Immunohistochemical stain for HCV core in the 2-month-old double transgenic mouse (DTM) liver with high (A, B), intermediate (C, D) and low (E, F) HCV core protein expression . The upper region of panels G, H and I depict results of Western blots for HCV core protein extracted from the same livers after two months on control chow (permissive diet). Detection in DTM with high (G), intermediate (H), and low HCV core protein expression (I). Lane 1, HeLa cells transfected with HCV core plasmid (positive control, He); lane 2, liver (li); lane 3, heart (h); lane 4, kidney (k); lane 5, thymus (t); lane 6, omental fat (o); lane 7, lung (lu); lane 8, muscle (m); lane 9, intestine (i); lane 10, skin (s). As a loading control, the same samples were probed for GAPDH (lower panel G, H, and I). (J) Quantitative immunoassays for hepatic HCV core protein from high, intermediate, and low protein-expressing mouse lines. Y-axis, core bands densities (units) acquired from Fluor-S multiimager.

Techniques Used: Immunohistochemistry, Staining, Transgenic Assay, Expressing, Western Blot, Transfection, Plasmid Preparation, Positive Control

7) Product Images from "The mitochondrial aspartate/glutamate carrier isoform 1 gene expression is regulated by CREB in neuronal cells"

Article Title: The mitochondrial aspartate/glutamate carrier isoform 1 gene expression is regulated by CREB in neuronal cells

Journal: The International Journal of Biochemistry & Cell Biology

doi: 10.1016/j.biocel.2015.01.004

Downregulation of CREB in cytokine-treated SH-SY5Y cells. P-CREB, T-CREB and β-actin proteins of SH-SY5Y cells treated or untreated with 500 U/m IFNγ, 1000 U/ml TNFα and 10 ng/ml IL-1β individually or in cambination for 24 h were immunodetected with specific antibodies. P-CREB blot was reprobed for T-CREB quantification. Similar results were obtained in four independent experiments. (B) The intensities of P-CREB and T-CREB in (A) were analyzed densitometrically using a Fluor-S MultiImager and Quantity One software (Bio-Rad) and corrected for β-actin. Data are expressed as means ± S.D. of four individual experiments. * P
Figure Legend Snippet: Downregulation of CREB in cytokine-treated SH-SY5Y cells. P-CREB, T-CREB and β-actin proteins of SH-SY5Y cells treated or untreated with 500 U/m IFNγ, 1000 U/ml TNFα and 10 ng/ml IL-1β individually or in cambination for 24 h were immunodetected with specific antibodies. P-CREB blot was reprobed for T-CREB quantification. Similar results were obtained in four independent experiments. (B) The intensities of P-CREB and T-CREB in (A) were analyzed densitometrically using a Fluor-S MultiImager and Quantity One software (Bio-Rad) and corrected for β-actin. Data are expressed as means ± S.D. of four individual experiments. * P

Techniques Used: Software

8) Product Images from "Molecular Characterization of Saccharomyces cerevisiae TFIID"

Article Title: Molecular Characterization of Saccharomyces cerevisiae TFIID

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.16.6000-6013.2002

Direct demonstration of TBP exchange. (A) Concentration dependence of TBP exchange within TFIID. Multiple, equal amounts of HA-TAF130p-tagged TFIID bound to protein A-Sepharose beads coated with anti-HA monoclonal antibody were generated as detailed in Materials and Methods. These aliquots of bead-bound TFIID were incubated with the indicated increasing molar excess of His 6 -tagged TBP for 30 min. Unbound His 6 -tagged TBP was removed by centrifugation and washing. The relative amounts of wild-type (WT) and His 6 -TBP remaining bead bound to TFIID were determined by SDS-PAGE and immunoblotting with polyclonal anti-TBP IgG. This immunoblot is shown in the inset and labeled WT TBP (TFIID-endogenous TBP) and His 6 -TBP (TBP exchanged into TFIID). Appropriate regions of the gel blot were separately probed with TFIID-specific antibodies (anti-TAF130p, anti-TAF65p, or anti-TAF48p IgGs). All immune complexes were detected by chemiluminescence and quantitated with a Fluor-S Bio-Rad MultiImager. Wild-type and His 6 -TBP content, corrected for average TAF recovery, is plotted as percent maximal signal as a function of His 6 -TBP/wild-type TBP ratio. Background subtracted and recovery-corrected wild-type TBP and His 6 -TBP values, in arbitrary units, were 123 and 0, 126 and 34, 116 and 76, 124 and 136, 92 and 126, 43 and 150, 27 and 161, and 21 and 149 in the reactions with His 6 -TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Average TAFp recoveries in these reactions ranged from 95 to 112%. (B) Kinetics of TBP-TFIID exchange. TBP-TFIID exchange was performed as above. Seven identical reactions were set up, each with a 10-fold molar excess of His 6 -tagged TBP relative to TFIID-endogenous wild-type TBP. Samples were incubated for 0, 10, 20, 30, 45, 60, or 120 min, as indicated. Wild-type and His 6 -TBP levels (upper) and TAFp content (lower) were determined by immunoblotting as in panel A. The levels of wild-type and His 6 -tagged TBP in these reactions were 75 and 0, 53 and 89, 51 and 92, 49 and 87, 38 and 96, 27 and 98, and 25 and 94 arbitrary units in the reactions with His 6 -TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Recovery of TAFs (lower) was in the same range as for panel A.
Figure Legend Snippet: Direct demonstration of TBP exchange. (A) Concentration dependence of TBP exchange within TFIID. Multiple, equal amounts of HA-TAF130p-tagged TFIID bound to protein A-Sepharose beads coated with anti-HA monoclonal antibody were generated as detailed in Materials and Methods. These aliquots of bead-bound TFIID were incubated with the indicated increasing molar excess of His 6 -tagged TBP for 30 min. Unbound His 6 -tagged TBP was removed by centrifugation and washing. The relative amounts of wild-type (WT) and His 6 -TBP remaining bead bound to TFIID were determined by SDS-PAGE and immunoblotting with polyclonal anti-TBP IgG. This immunoblot is shown in the inset and labeled WT TBP (TFIID-endogenous TBP) and His 6 -TBP (TBP exchanged into TFIID). Appropriate regions of the gel blot were separately probed with TFIID-specific antibodies (anti-TAF130p, anti-TAF65p, or anti-TAF48p IgGs). All immune complexes were detected by chemiluminescence and quantitated with a Fluor-S Bio-Rad MultiImager. Wild-type and His 6 -TBP content, corrected for average TAF recovery, is plotted as percent maximal signal as a function of His 6 -TBP/wild-type TBP ratio. Background subtracted and recovery-corrected wild-type TBP and His 6 -TBP values, in arbitrary units, were 123 and 0, 126 and 34, 116 and 76, 124 and 136, 92 and 126, 43 and 150, 27 and 161, and 21 and 149 in the reactions with His 6 -TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Average TAFp recoveries in these reactions ranged from 95 to 112%. (B) Kinetics of TBP-TFIID exchange. TBP-TFIID exchange was performed as above. Seven identical reactions were set up, each with a 10-fold molar excess of His 6 -tagged TBP relative to TFIID-endogenous wild-type TBP. Samples were incubated for 0, 10, 20, 30, 45, 60, or 120 min, as indicated. Wild-type and His 6 -TBP levels (upper) and TAFp content (lower) were determined by immunoblotting as in panel A. The levels of wild-type and His 6 -tagged TBP in these reactions were 75 and 0, 53 and 89, 51 and 92, 49 and 87, 38 and 96, 27 and 98, and 25 and 94 arbitrary units in the reactions with His 6 -TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Recovery of TAFs (lower) was in the same range as for panel A.

Techniques Used: Concentration Assay, Generated, Incubation, Centrifugation, SDS Page, Labeling, Western Blot

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Mass Spectrometry:

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Article Snippet: Probes were synthesized using a random primed DNA labeling kit (Roche Molecular Biochemicals, Indianapolis), the 3′-untranslated region of the TBG cDNAs as a template, and [α-32 P]dATP (3,000 Ci mmol−1 ). .. Autoradiographic data were quantified using a Fluor-S MultiImager and Quantity One software (Bio-Rad, Hercules, CA).

Autoradiography:

Article Title: SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1 [W]
Article Snippet: .. The intensity of each band in the autoradiograph was quantified by the Fluor-S-MultiImager (Bio-Rad) and ratios of the CAB1 or RBCS gene versus its corresponding 18S ribosomal RNA (rRNA) band were determined and plotted (Fluor-S-MultiImager; Bio-Rad). ..

Construct:

Article Title: Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation
Article Snippet: HeLa cells carrying the same LAP-tTA (Clontech, Mountain View, CA) were transiently transfected with the TRE-HCV core construct in the absence of DOX. .. HCV core protein band intensity was quantified using a Fluor-S multiimager and Quality One software (Bio-Rad).

Electrophoresis:

Article Title: Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation
Article Snippet: After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) and incubated with a 1:250 dilution of monoclonal mouse anti-HCV core antibody (Anogen, Mississauga, Ontario, Canada). .. HCV core protein band intensity was quantified using a Fluor-S multiimager and Quality One software (Bio-Rad).

Article Title: Molecular and Cellular Characterization of the Tomato Pollen Profilin, LePro1
Article Snippet: Ten micrograms of proteins per sample were loaded onto 14% SDS-polyacrylamide gel and separated by electrophoresis. .. Protein signals were scanned with Fluor-S™ multiImager (Bio-Rad) and quantified by the unit density of pixels using the enclosed Quantity 1 software.

Incubation:

Article Title: Molecular Characterization of Saccharomyces cerevisiae TFIID
Article Snippet: To control for any effects of either incubation time or dilution, a mock reaction was performed by diluting 50 μl of WCE with 150 μl of dilution buffer and incubating it simultaneously. .. To estimate TBP concentrations and the extent of TFIID depletion, immunoblots were developed with Lumi-Light Plus Western blotting substrate (Roche) and quantitated with a Fluor-S MultiImager (Bio-Rad).

Article Title: Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation
Article Snippet: After washes, membranes were incubated with secondary antibody (Bio-Rad), and developed using an ECL kit (Amersham, Buckinghamshire, UK). .. HCV core protein band intensity was quantified using a Fluor-S multiimager and Quality One software (Bio-Rad).

Article Title: The mitochondrial aspartate/glutamate carrier isoform 1 gene expression is regulated by CREB in neuronal cells
Article Snippet: Band intensities were analyzed densitometrically using a Fluor-S MultiImager and Quantity One software (Bio-Rad). .. In brief, cells were seeded onto 96-well microtiter plates and treated with siRNA targeting CREB (siCREB) for up to 72 h. After incubation, 15 μl of the Dye Solution was added to each well and the cells were incubated for 4 h at 37 °C.

Article Title: Molecular and Cellular Characterization of the Tomato Pollen Profilin, LePro1
Article Snippet: The membranes were then incubated with polyclonal anti-tomato profilin antibody and monoclonal anti-pea actin antibody for one hour. .. Protein signals were scanned with Fluor-S™ multiImager (Bio-Rad) and quantified by the unit density of pixels using the enclosed Quantity 1 software.

Proliferation Assay:

Article Title: The mitochondrial aspartate/glutamate carrier isoform 1 gene expression is regulated by CREB in neuronal cells
Article Snippet: Band intensities were analyzed densitometrically using a Fluor-S MultiImager and Quantity One software (Bio-Rad). .. Cell viability was evaluated by the CellTiter 96® Non-Radioactive Cell Proliferation Assay (Promega).

Expressing:

Article Title: Skp2 Regulates G2/M Progression in a p53-dependent Manner
Article Snippet: Melanoma cell lysates were analyzed for protein expression by Western blotting as described previously ( ). .. Western blots were developed using SuperSignal chemiluminescent substrate (Pierce Chemical, Rockford) and quantitated with a Fluor-S MultiImager and Quantity-One software (Bio-Rad, Hercules, CA).

Article Title: Antenatal Corticosteroids and Postnatal Fluid Restriction Produce Differential Effects on AQP3 Expression, Water Handling, and Barrier Function in Perinatal Rat Epidermis
Article Snippet: Paragraph title: 2.5. Semiquantitative Analysis of AQP3 mRNA Expression ... Digital images were acquired with use of a Fluor-S MultiImager and analyzed with the original software (Quantity One, version 4.2.1, Bio-Rad Laboratories, Hercules, CA, USA) after subtraction of matched backgrounds.

Western Blot:

Article Title: Molecular Characterization of Saccharomyces cerevisiae TFIID
Article Snippet: .. To estimate TBP concentrations and the extent of TFIID depletion, immunoblots were developed with Lumi-Light Plus Western blotting substrate (Roche) and quantitated with a Fluor-S MultiImager (Bio-Rad). .. The adenovirus type 2 (Ad2) major late promoter (MLP) footprinting probe, containing sequences from −250 to +195 (numbering relative to the +1 transcription start site), was generated by excising an Eco RI/ Hin dIII fragment from plasmid pML4 ( ).

Article Title: Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation
Article Snippet: HeLa cell or mouse liver protein extracts were analyzed by Western blotting. .. HCV core protein band intensity was quantified using a Fluor-S multiimager and Quality One software (Bio-Rad).

Article Title: Skp2 Regulates G2/M Progression in a p53-dependent Manner
Article Snippet: .. Western blots were developed using SuperSignal chemiluminescent substrate (Pierce Chemical, Rockford) and quantitated with a Fluor-S MultiImager and Quantity-One software (Bio-Rad, Hercules, CA). ..

Article Title: Down-Regulation of Tomato ?-Galactosidase 4 Results in Decreased Fruit Softening 1
Article Snippet: Paragraph title: RNA Extraction and Gel-Blot Analysis ... Autoradiographic data were quantified using a Fluor-S MultiImager and Quantity One software (Bio-Rad, Hercules, CA).

Hybridization:

Article Title: Down-Regulation of Tomato ?-Galactosidase 4 Results in Decreased Fruit Softening 1
Article Snippet: As a loading control, RNA blots were stripped and reprobed at a reduced hybridization and washing stringency using a soybean ( Glycine max ) 26S rDNA fragment. .. Autoradiographic data were quantified using a Fluor-S MultiImager and Quantity One software (Bio-Rad, Hercules, CA).

Transfection:

Article Title: Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation
Article Snippet: HeLa cells carrying the same LAP-tTA (Clontech, Mountain View, CA) were transiently transfected with the TRE-HCV core construct in the absence of DOX. .. HCV core protein band intensity was quantified using a Fluor-S multiimager and Quality One software (Bio-Rad).

Sequencing:

Article Title: Inhibition of a Secreted Glutamic Peptidase Prevents Growth of the FungusTalaromyces emersonii *
Article Snippet: The complete coding sequence of tgp1 was confirmed by PCR amplification and sequencing of cDNA using gene-specific oligonucleotides (5′-CAGGTCATTCCCAAGATCA-3′ and 5′-CAAGATATTCTTCTCAACA-3′). .. Images were visualized using a Fluor-S™ MultiImager (Bio-Rad).

Northern Blot:

Article Title: SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1 [W]
Article Snippet: Paragraph title: Northern-Blot Analyses ... The intensity of each band in the autoradiograph was quantified by the Fluor-S-MultiImager (Bio-Rad) and ratios of the CAB1 or RBCS gene versus its corresponding 18S ribosomal RNA (rRNA) band were determined and plotted (Fluor-S-MultiImager; Bio-Rad).

Article Title: Inhibition of a Secreted Glutamic Peptidase Prevents Growth of the FungusTalaromyces emersonii *
Article Snippet: Northern blots were performed on total RNA as outlined previously , using the digoxigenin-labeled tgp1 probe. .. Images were visualized using a Fluor-S™ MultiImager (Bio-Rad).

DNA Labeling:

Article Title: Down-Regulation of Tomato ?-Galactosidase 4 Results in Decreased Fruit Softening 1
Article Snippet: Probes were synthesized using a random primed DNA labeling kit (Roche Molecular Biochemicals, Indianapolis), the 3′-untranslated region of the TBG cDNAs as a template, and [α-32 P]dATP (3,000 Ci mmol−1 ). .. Autoradiographic data were quantified using a Fluor-S MultiImager and Quantity One software (Bio-Rad, Hercules, CA).

Polymerase Chain Reaction:

Article Title: Inhibition of a Secreted Glutamic Peptidase Prevents Growth of the FungusTalaromyces emersonii *
Article Snippet: The complete coding sequence of tgp1 was confirmed by PCR amplification and sequencing of cDNA using gene-specific oligonucleotides (5′-CAGGTCATTCCCAAGATCA-3′ and 5′-CAAGATATTCTTCTCAACA-3′). .. Images were visualized using a Fluor-S™ MultiImager (Bio-Rad).

Article Title: Smurf2 E3 ubiquitin ligase modulates proliferation and invasiveness of breast cancer cells in a CNKSR2 dependent manner
Article Snippet: Paragraph title: Reverse transcriptase -PCR ... Analysis of amplified products was done on 2% agarose gel and visualized using Fluor-S™ MultiImager (Bio-Rad).

Article Title: Antenatal Corticosteroids and Postnatal Fluid Restriction Produce Differential Effects on AQP3 Expression, Water Handling, and Barrier Function in Perinatal Rat Epidermis
Article Snippet: GeneRuler 100 bp DNA ladder (Fermentas, Vilnius, Lithuania) was used for sizing of PCR fragments. .. Digital images were acquired with use of a Fluor-S MultiImager and analyzed with the original software (Quantity One, version 4.2.1, Bio-Rad Laboratories, Hercules, CA, USA) after subtraction of matched backgrounds.

Pulsed-Field Gel:

Article Title: Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in Z?rich, Switzerland (2003): Prevalence of Type IV SCCmec and a New SCCmec Element Associated with Isolates from Intravenous Drug Users
Article Snippet: The MRSAs were genotyped by pulsed-field gel electrophoresis (PFGE) of SmaI-digested chromosomal DNA, following the protocol of Wada et al. ( ). .. The banding patterns were analyzed visually, by scanning with a Fluor-S MultiImager, and by digital analysis with a Multianalys/PC (Bio-Rad) ( ).

Nucleic Acid Electrophoresis:

Article Title: Molecular Characterization of Saccharomyces cerevisiae TFIID
Article Snippet: Purified TFIID was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) on a 10% NuPAGE gel with MOPS (morpholinepropanesulfonic acid) running buffer (Invitrogen). .. Gels were stained with either Coomassie brilliant blue R-250 (Sigma) or SYPRO Ruby (Molecular Probes) ( ) and imaged with a Fluor-S MultiImager (Bio-Rad).

Isolation:

Article Title: Inhibition of a Secreted Glutamic Peptidase Prevents Growth of the FungusTalaromyces emersonii *
Article Snippet: RNA Extraction and Northern Blot Analysis —Total RNA was isolated from T. emersonii with TRI-Reagent (Molecular Research Centre). .. Images were visualized using a Fluor-S™ MultiImager (Bio-Rad).

Article Title: Smurf2 E3 ubiquitin ligase modulates proliferation and invasiveness of breast cancer cells in a CNKSR2 dependent manner
Article Snippet: Reverse transcriptase -PCR Total RNA was isolated using TRIzol (Invitrogen) reagent according to the manufacturer’s protocol. .. Analysis of amplified products was done on 2% agarose gel and visualized using Fluor-S™ MultiImager (Bio-Rad).

Protein Concentration:

Article Title: Molecular and Cellular Characterization of the Tomato Pollen Profilin, LePro1
Article Snippet: Protein concentration was determined using the BioRad protein assay kit (BioRad, CA). .. Protein signals were scanned with Fluor-S™ multiImager (Bio-Rad) and quantified by the unit density of pixels using the enclosed Quantity 1 software.

Immunohistochemistry:

Article Title: Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation
Article Snippet: HCV core protein band intensity was quantified using a Fluor-S multiimager and Quality One software (Bio-Rad). .. For immunohistochemical analysis, tissue samples were harvested, fixed in 4% paraformaldehyde, treated with 0.1% Triton X-100, incubated with HCV core protein antibody, and subsequently incubated with secondary antibody (Caltag, Carlsbad, CA).

Labeling:

Article Title: SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1 [W]
Article Snippet: The 0.6-kb SHW1 DNA fragment was used as probe after random prime labeling (Amersham). .. The intensity of each band in the autoradiograph was quantified by the Fluor-S-MultiImager (Bio-Rad) and ratios of the CAB1 or RBCS gene versus its corresponding 18S ribosomal RNA (rRNA) band were determined and plotted (Fluor-S-MultiImager; Bio-Rad).

Purification:

Article Title: Molecular Characterization of Saccharomyces cerevisiae TFIID
Article Snippet: Purified TFIID was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) on a 10% NuPAGE gel with MOPS (morpholinepropanesulfonic acid) running buffer (Invitrogen). .. Gels were stained with either Coomassie brilliant blue R-250 (Sigma) or SYPRO Ruby (Molecular Probes) ( ) and imaged with a Fluor-S MultiImager (Bio-Rad).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Smurf2 E3 ubiquitin ligase modulates proliferation and invasiveness of breast cancer cells in a CNKSR2 dependent manner
Article Snippet: The RT-PCR reaction mixture contained 5 μl of 10× reaction buffer, 2 μl of cDNA template, 1 μl each of forward and reverse primers, and 0.5 μl of Taq DNA polymerase (Sigma) in a final volume of 20 μl. .. Analysis of amplified products was done on 2% agarose gel and visualized using Fluor-S™ MultiImager (Bio-Rad).

Protein Extraction:

Article Title: Molecular and Cellular Characterization of the Tomato Pollen Profilin, LePro1
Article Snippet: Paragraph title: Protein Extraction and Immunoblotting ... Protein signals were scanned with Fluor-S™ multiImager (Bio-Rad) and quantified by the unit density of pixels using the enclosed Quantity 1 software.

Polyacrylamide Gel Electrophoresis:

Article Title: Molecular Characterization of Saccharomyces cerevisiae TFIID
Article Snippet: Purified TFIID was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) on a 10% NuPAGE gel with MOPS (morpholinepropanesulfonic acid) running buffer (Invitrogen). .. Gels were stained with either Coomassie brilliant blue R-250 (Sigma) or SYPRO Ruby (Molecular Probes) ( ) and imaged with a Fluor-S MultiImager (Bio-Rad).

Random Primed:

Article Title: Down-Regulation of Tomato ?-Galactosidase 4 Results in Decreased Fruit Softening 1
Article Snippet: Probes were synthesized using a random primed DNA labeling kit (Roche Molecular Biochemicals, Indianapolis), the 3′-untranslated region of the TBG cDNAs as a template, and [α-32 P]dATP (3,000 Ci mmol−1 ). .. Autoradiographic data were quantified using a Fluor-S MultiImager and Quantity One software (Bio-Rad, Hercules, CA).

Plasmid Preparation:

Article Title: Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation
Article Snippet: HCV core protein band intensity was quantified using a Fluor-S multiimager and Quality One software (Bio-Rad). .. The background staining was eliminated by a mouse-on-mouse kit (Vector, Burlingame, CA).

Software:

Article Title: Itinerary of Hepatitis B Viruses: Delineation of Restriction Points Critical for Infectious Entry
Article Snippet: .. Signals were quantified after acquisition with a Fluor-S MultiImager by using Quantity One software. .. As a first step towards delineation of the nature and number of cellular factors restricting hepadnavirus infection in vitro, we analyzed viral attachment and entry kinetics by using DHBV-permissive PDHs.

Article Title: Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation
Article Snippet: .. HCV core protein band intensity was quantified using a Fluor-S multiimager and Quality One software (Bio-Rad). .. Glyceraldehyde-3-phosphate dehydrogenase (Abcam, Cambridge, UK) was used as an internal control.

Article Title: The mitochondrial aspartate/glutamate carrier isoform 1 gene expression is regulated by CREB in neuronal cells
Article Snippet: .. Band intensities were analyzed densitometrically using a Fluor-S MultiImager and Quantity One software (Bio-Rad). .. Cell viability was evaluated by the CellTiter 96® Non-Radioactive Cell Proliferation Assay (Promega).

Article Title: Skp2 Regulates G2/M Progression in a p53-dependent Manner
Article Snippet: .. Western blots were developed using SuperSignal chemiluminescent substrate (Pierce Chemical, Rockford) and quantitated with a Fluor-S MultiImager and Quantity-One software (Bio-Rad, Hercules, CA). ..

Article Title: Smurf2 E3 ubiquitin ligase modulates proliferation and invasiveness of breast cancer cells in a CNKSR2 dependent manner
Article Snippet: Analysis of amplified products was done on 2% agarose gel and visualized using Fluor-S™ MultiImager (Bio-Rad). .. The PCR products were quantified by densitometric analysis, using Bio-Rad Quantity One software.

Article Title: Down-Regulation of Tomato ?-Galactosidase 4 Results in Decreased Fruit Softening 1
Article Snippet: .. Autoradiographic data were quantified using a Fluor-S MultiImager and Quantity One software (Bio-Rad, Hercules, CA). .. To analyze the accumulation of other TBG mRNAs (Fig. ), a more sensitive RNA gel-blot method was used.

Article Title: Molecular and Cellular Characterization of the Tomato Pollen Profilin, LePro1
Article Snippet: .. Protein signals were scanned with Fluor-S™ multiImager (Bio-Rad) and quantified by the unit density of pixels using the enclosed Quantity 1 software. .. Pollen Germination and Morphological Analysis For in vitro pollen germination, pollen grains were collected as described previously and suspended in tomato pollen germination medium (TPGM) containing 10% sucrose, 0.01% KNO3 , 0.01% H3 BO3 , 0.02% MgSO4 , 0.06% Ca(NO3 )2 and 20 mM MES (pH 7.0).

Article Title: Antenatal Corticosteroids and Postnatal Fluid Restriction Produce Differential Effects on AQP3 Expression, Water Handling, and Barrier Function in Perinatal Rat Epidermis
Article Snippet: .. Digital images were acquired with use of a Fluor-S MultiImager and analyzed with the original software (Quantity One, version 4.2.1, Bio-Rad Laboratories, Hercules, CA, USA) after subtraction of matched backgrounds. ..

RNA Extraction:

Article Title: Inhibition of a Secreted Glutamic Peptidase Prevents Growth of the FungusTalaromyces emersonii *
Article Snippet: RNA Extraction and Northern Blot Analysis —Total RNA was isolated from T. emersonii with TRI-Reagent (Molecular Research Centre). .. Images were visualized using a Fluor-S™ MultiImager (Bio-Rad).

Article Title: Down-Regulation of Tomato ?-Galactosidase 4 Results in Decreased Fruit Softening 1
Article Snippet: Paragraph title: RNA Extraction and Gel-Blot Analysis ... Autoradiographic data were quantified using a Fluor-S MultiImager and Quantity One software (Bio-Rad, Hercules, CA).

Agarose Gel Electrophoresis:

Article Title: Smurf2 E3 ubiquitin ligase modulates proliferation and invasiveness of breast cancer cells in a CNKSR2 dependent manner
Article Snippet: .. Analysis of amplified products was done on 2% agarose gel and visualized using Fluor-S™ MultiImager (Bio-Rad). .. The PCR products were quantified by densitometric analysis, using Bio-Rad Quantity One software.

Article Title: Antenatal Corticosteroids and Postnatal Fluid Restriction Produce Differential Effects on AQP3 Expression, Water Handling, and Barrier Function in Perinatal Rat Epidermis
Article Snippet: All individual PCR products were run simultaneously on 1.5% agarose gel with 1X TAE buffer, containing 0.5 μ g/mL GelStar stain (BioWhittaker Molecular Applications, Rockland, ME, USA). .. Digital images were acquired with use of a Fluor-S MultiImager and analyzed with the original software (Quantity One, version 4.2.1, Bio-Rad Laboratories, Hercules, CA, USA) after subtraction of matched backgrounds.

In Vitro:

Article Title: Molecular Characterization of Saccharomyces cerevisiae TFIID
Article Snippet: Paragraph title: In vitro transcription. ... To estimate TBP concentrations and the extent of TFIID depletion, immunoblots were developed with Lumi-Light Plus Western blotting substrate (Roche) and quantitated with a Fluor-S MultiImager (Bio-Rad).

Concentration Assay:

Article Title: Antenatal Corticosteroids and Postnatal Fluid Restriction Produce Differential Effects on AQP3 Expression, Water Handling, and Barrier Function in Perinatal Rat Epidermis
Article Snippet: Digital images were acquired with use of a Fluor-S MultiImager and analyzed with the original software (Quantity One, version 4.2.1, Bio-Rad Laboratories, Hercules, CA, USA) after subtraction of matched backgrounds. .. The level of 18S rRNA expression was measured in all samples and used to normalize the AQP expression, thus correcting for any sample-to-sample differences in RNA concentration, RNA quality, and efficiency of the RT and PCR reactions.

Thin Layer Chromatography:

Article Title: Glycosphingolipids Promote Entry of a Broad Range of Human Immunodeficiency Virus Type 1 Isolates into Cell Lines Expressing CD4, CXCR4, and/or CCR5
Article Snippet: The total GSL composition of cells before and after treatment with PPMP was analyzed by thin-layer chromatography (TLC) developed in CHCl3 –CH3 OH–10% KCl(aq) (50:40:10, vol/vol/vol). .. At the end of the run, the plate was air dried, sprayed with resorcinol , heated at 100°C for 20 min to develop the spots, and scanned with a Fluor-S MultiImager (Bio-Rad, Hercules, Calif.).

In Situ:

Article Title: Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation
Article Snippet: Paragraph title: Evaluation of the in situ HCV core protein level ... HCV core protein band intensity was quantified using a Fluor-S multiimager and Quality One software (Bio-Rad).

Staining:

Article Title: Hepatic inflammation mediated by hepatitis C virus core protein is ameliorated by blocking complement activation
Article Snippet: HCV core protein band intensity was quantified using a Fluor-S multiimager and Quality One software (Bio-Rad). .. The background staining was eliminated by a mouse-on-mouse kit (Vector, Burlingame, CA).

Article Title: Down-Regulation of Tomato ?-Galactosidase 4 Results in Decreased Fruit Softening 1
Article Snippet: The membranes were then washed several times with 0.2× SSC with 0.5% (w/v) SDS to remove the methylene blue stain. .. Autoradiographic data were quantified using a Fluor-S MultiImager and Quantity One software (Bio-Rad, Hercules, CA).

Article Title: Molecular Characterization of Saccharomyces cerevisiae TFIID
Article Snippet: .. Gels were stained with either Coomassie brilliant blue R-250 (Sigma) or SYPRO Ruby (Molecular Probes) ( ) and imaged with a Fluor-S MultiImager (Bio-Rad). ..

Article Title: Antenatal Corticosteroids and Postnatal Fluid Restriction Produce Differential Effects on AQP3 Expression, Water Handling, and Barrier Function in Perinatal Rat Epidermis
Article Snippet: All individual PCR products were run simultaneously on 1.5% agarose gel with 1X TAE buffer, containing 0.5 μ g/mL GelStar stain (BioWhittaker Molecular Applications, Rockland, ME, USA). .. Digital images were acquired with use of a Fluor-S MultiImager and analyzed with the original software (Quantity One, version 4.2.1, Bio-Rad Laboratories, Hercules, CA, USA) after subtraction of matched backgrounds.

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    Bio-Rad fluor s max multiimager
    A fraction of hsMOK2 protein is associated with nuclear matrix. HeLa cells transfected with CMV-hsMOK2 ( A ), CMV-hsMOK2Δ ( B ) or CMV-NH 2 hsMOK2 ( C ) vectors were sequentially extracted as described in Materials and Methods. The various protein fractions correspond to 1% Triton X-100 (fraction 1), DNase I at 37°C and 0.25 M (NH 4 ) 2 SO 4 (fraction 2), 2 M NaCl (fraction 3), RNase A (fraction 4) and, finally, the pellet containing nuclear matrix (fraction 5) resuspended in SDS sample buffer. Four micrograms of proteins from fractions 1–4 and 20 µg of proteins from fraction 5, corresponding to the nuclear matrix, were subjected to SDS–PAGE and western blot analysis. Probing was realized with polyclonal anti-hsMOK2 antibody to detect hsMOK2 and truncated proteins or monoclonal anti-lamin A/C(636) antibody to detect the two nuclear matrix lamins A and C. Western blots were visualized with a <t>Fluor-S</t> Max <t>MultiImager</t> and quantified with Quantity One software (Bio-Rad). The percentages indicated below each panel were normalized at 4 µg of proteins. The molecular weights of the proteins are indicated on the right.
    Fluor S Max Multiimager, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A fraction of hsMOK2 protein is associated with nuclear matrix. HeLa cells transfected with CMV-hsMOK2 ( A ), CMV-hsMOK2Δ ( B ) or CMV-NH 2 hsMOK2 ( C ) vectors were sequentially extracted as described in Materials and Methods. The various protein fractions correspond to 1% Triton X-100 (fraction 1), DNase I at 37°C and 0.25 M (NH 4 ) 2 SO 4 (fraction 2), 2 M NaCl (fraction 3), RNase A (fraction 4) and, finally, the pellet containing nuclear matrix (fraction 5) resuspended in SDS sample buffer. Four micrograms of proteins from fractions 1–4 and 20 µg of proteins from fraction 5, corresponding to the nuclear matrix, were subjected to SDS–PAGE and western blot analysis. Probing was realized with polyclonal anti-hsMOK2 antibody to detect hsMOK2 and truncated proteins or monoclonal anti-lamin A/C(636) antibody to detect the two nuclear matrix lamins A and C. Western blots were visualized with a Fluor-S Max MultiImager and quantified with Quantity One software (Bio-Rad). The percentages indicated below each panel were normalized at 4 µg of proteins. The molecular weights of the proteins are indicated on the right.

    Journal: Nucleic Acids Research

    Article Title: In vivo and in vitro interaction between human transcription factor MOK2 and nuclear lamin A/C

    doi:

    Figure Lengend Snippet: A fraction of hsMOK2 protein is associated with nuclear matrix. HeLa cells transfected with CMV-hsMOK2 ( A ), CMV-hsMOK2Δ ( B ) or CMV-NH 2 hsMOK2 ( C ) vectors were sequentially extracted as described in Materials and Methods. The various protein fractions correspond to 1% Triton X-100 (fraction 1), DNase I at 37°C and 0.25 M (NH 4 ) 2 SO 4 (fraction 2), 2 M NaCl (fraction 3), RNase A (fraction 4) and, finally, the pellet containing nuclear matrix (fraction 5) resuspended in SDS sample buffer. Four micrograms of proteins from fractions 1–4 and 20 µg of proteins from fraction 5, corresponding to the nuclear matrix, were subjected to SDS–PAGE and western blot analysis. Probing was realized with polyclonal anti-hsMOK2 antibody to detect hsMOK2 and truncated proteins or monoclonal anti-lamin A/C(636) antibody to detect the two nuclear matrix lamins A and C. Western blots were visualized with a Fluor-S Max MultiImager and quantified with Quantity One software (Bio-Rad). The percentages indicated below each panel were normalized at 4 µg of proteins. The molecular weights of the proteins are indicated on the right.

    Article Snippet: The blots were visualized using a Fluor-S Max MultiImager with Quantity One software (Bio-Rad).

    Techniques: Transfection, SDS Page, Western Blot, Software

    Direct demonstration of TBP exchange. (A) Concentration dependence of TBP exchange within TFIID. Multiple, equal amounts of HA-TAF130p-tagged TFIID bound to protein A-Sepharose beads coated with anti-HA monoclonal antibody were generated as detailed in Materials and Methods. These aliquots of bead-bound TFIID were incubated with the indicated increasing molar excess of His 6 -tagged TBP for 30 min. Unbound His 6 -tagged TBP was removed by centrifugation and washing. The relative amounts of wild-type (WT) and His 6 -TBP remaining bead bound to TFIID were determined by SDS-PAGE and immunoblotting with polyclonal anti-TBP IgG. This immunoblot is shown in the inset and labeled WT TBP (TFIID-endogenous TBP) and His 6 -TBP (TBP exchanged into TFIID). Appropriate regions of the gel blot were separately probed with TFIID-specific antibodies (anti-TAF130p, anti-TAF65p, or anti-TAF48p IgGs). All immune complexes were detected by chemiluminescence and quantitated with a Fluor-S Bio-Rad MultiImager. Wild-type and His 6 -TBP content, corrected for average TAF recovery, is plotted as percent maximal signal as a function of His 6 -TBP/wild-type TBP ratio. Background subtracted and recovery-corrected wild-type TBP and His 6 -TBP values, in arbitrary units, were 123 and 0, 126 and 34, 116 and 76, 124 and 136, 92 and 126, 43 and 150, 27 and 161, and 21 and 149 in the reactions with His 6 -TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Average TAFp recoveries in these reactions ranged from 95 to 112%. (B) Kinetics of TBP-TFIID exchange. TBP-TFIID exchange was performed as above. Seven identical reactions were set up, each with a 10-fold molar excess of His 6 -tagged TBP relative to TFIID-endogenous wild-type TBP. Samples were incubated for 0, 10, 20, 30, 45, 60, or 120 min, as indicated. Wild-type and His 6 -TBP levels (upper) and TAFp content (lower) were determined by immunoblotting as in panel A. The levels of wild-type and His 6 -tagged TBP in these reactions were 75 and 0, 53 and 89, 51 and 92, 49 and 87, 38 and 96, 27 and 98, and 25 and 94 arbitrary units in the reactions with His 6 -TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Recovery of TAFs (lower) was in the same range as for panel A.

    Journal: Molecular and Cellular Biology

    Article Title: Molecular Characterization of Saccharomyces cerevisiae TFIID

    doi: 10.1128/MCB.22.16.6000-6013.2002

    Figure Lengend Snippet: Direct demonstration of TBP exchange. (A) Concentration dependence of TBP exchange within TFIID. Multiple, equal amounts of HA-TAF130p-tagged TFIID bound to protein A-Sepharose beads coated with anti-HA monoclonal antibody were generated as detailed in Materials and Methods. These aliquots of bead-bound TFIID were incubated with the indicated increasing molar excess of His 6 -tagged TBP for 30 min. Unbound His 6 -tagged TBP was removed by centrifugation and washing. The relative amounts of wild-type (WT) and His 6 -TBP remaining bead bound to TFIID were determined by SDS-PAGE and immunoblotting with polyclonal anti-TBP IgG. This immunoblot is shown in the inset and labeled WT TBP (TFIID-endogenous TBP) and His 6 -TBP (TBP exchanged into TFIID). Appropriate regions of the gel blot were separately probed with TFIID-specific antibodies (anti-TAF130p, anti-TAF65p, or anti-TAF48p IgGs). All immune complexes were detected by chemiluminescence and quantitated with a Fluor-S Bio-Rad MultiImager. Wild-type and His 6 -TBP content, corrected for average TAF recovery, is plotted as percent maximal signal as a function of His 6 -TBP/wild-type TBP ratio. Background subtracted and recovery-corrected wild-type TBP and His 6 -TBP values, in arbitrary units, were 123 and 0, 126 and 34, 116 and 76, 124 and 136, 92 and 126, 43 and 150, 27 and 161, and 21 and 149 in the reactions with His 6 -TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Average TAFp recoveries in these reactions ranged from 95 to 112%. (B) Kinetics of TBP-TFIID exchange. TBP-TFIID exchange was performed as above. Seven identical reactions were set up, each with a 10-fold molar excess of His 6 -tagged TBP relative to TFIID-endogenous wild-type TBP. Samples were incubated for 0, 10, 20, 30, 45, 60, or 120 min, as indicated. Wild-type and His 6 -TBP levels (upper) and TAFp content (lower) were determined by immunoblotting as in panel A. The levels of wild-type and His 6 -tagged TBP in these reactions were 75 and 0, 53 and 89, 51 and 92, 49 and 87, 38 and 96, 27 and 98, and 25 and 94 arbitrary units in the reactions with His 6 -TBP/wild-type TBP ratios of 0, 1, 3, 5, 10, 30, 60, and 100, respectively. Recovery of TAFs (lower) was in the same range as for panel A.

    Article Snippet: Gels were stained with either Coomassie brilliant blue R-250 (Sigma) or SYPRO Ruby (Molecular Probes) ( ) and imaged with a Fluor-S MultiImager (Bio-Rad).

    Techniques: Concentration Assay, Generated, Incubation, Centrifugation, SDS Page, Labeling, Western Blot

    Chlorophyll and anthocyanin contents and the expression of light-inducible genes in shw1 mutants. A, Accumulation of chlorophyll in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings. B, Accumulation of anthocyanin in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings. C, Quantification of RNA gel-blot analysis data of CAB1 in wild type (Col) and shw1-1 mutant seedlings. Five-day-old dark-grown seedlings were transferred to WL for 2, 4, and 8 h. Five-day-old dark-grown seedlings have been shown as 0 h. The experiment was repeated three times. The intensity of each band in autoradiograph was quantified by the Fluor-S-MultiImager (Bio-Rad) and ratios of the CAB1 gene versus its corresponding 18S rRNA band were determined and plotted (Fluor-S-MultiImager; Bio-Rad). D, Quantification of RNA gel-blot analysis data of RBCS in wild type (Col) and shw1-1 mutant seedlings. For experimental detail see legend to Figure 5C. E, Accumulation of chlorophyll in the cotyledons after normalized by cotyledon size in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings.

    Journal: Plant Physiology

    Article Title: SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1SHORT HYPOCOTYL IN WHITE LIGHT1, a Serine-Arginine-Aspartate-Rich Protein in Arabidopsis, Acts as a Negative Regulator of Photomorphogenic Growth 1 [W]

    doi: 10.1104/pp.108.118174

    Figure Lengend Snippet: Chlorophyll and anthocyanin contents and the expression of light-inducible genes in shw1 mutants. A, Accumulation of chlorophyll in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings. B, Accumulation of anthocyanin in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings. C, Quantification of RNA gel-blot analysis data of CAB1 in wild type (Col) and shw1-1 mutant seedlings. Five-day-old dark-grown seedlings were transferred to WL for 2, 4, and 8 h. Five-day-old dark-grown seedlings have been shown as 0 h. The experiment was repeated three times. The intensity of each band in autoradiograph was quantified by the Fluor-S-MultiImager (Bio-Rad) and ratios of the CAB1 gene versus its corresponding 18S rRNA band were determined and plotted (Fluor-S-MultiImager; Bio-Rad). D, Quantification of RNA gel-blot analysis data of RBCS in wild type (Col) and shw1-1 mutant seedlings. For experimental detail see legend to Figure 5C. E, Accumulation of chlorophyll in the cotyledons after normalized by cotyledon size in 6-d-old constant WL (30 μ mol m −2 s −1 ) grown wild type (Col) and shw1-1 mutant seedlings.

    Article Snippet: The intensity of each band in the autoradiograph was quantified by the Fluor-S-MultiImager (Bio-Rad) and ratios of the CAB1 or RBCS gene versus its corresponding 18S ribosomal RNA (rRNA) band were determined and plotted (Fluor-S-MultiImager; Bio-Rad).

    Techniques: Expressing, Mutagenesis, Western Blot, Autoradiography

    Relative binding affinities of p53 and ΔNp63α for p53 consensus sites in the p21 and 14-3-3σ promoters. (A) The p53 consensus binding sequence and p53 binding sites in the p21 and 14-3-3σ promoters. Abbreviations: R, purine; Y, pyrimidine; W, adenine or thymine. (B) H1299 cells were transfected with myc-tagged p53 or ΔNp63α, and protein lysates were quantified by using the Fluor-S Max MultiImager. Based on Fluor-S Max quantification, equal amounts of myc-tagged p53 and myc-tagged ΔNp63α were immunoprecipitated with a myc epitope antibody. Immunoprecipitated p53 and ΔNp63α were assayed for their ability to bind radiolabeled oligonucleotides representing p53-binding sites in the p21 and 14-3-3σ promoters as described in Materials and Methods. (C) Bound oligonucleotides were separated on acrylamide gels, exposed for autoradiography, and quantified. Each autoradiograph shows one representative result of at least three independent experiments that are quantified and displayed with the standard deviation.

    Journal: Molecular and Cellular Biology

    Article Title: The ?Np63? Phosphoprotein Binds the p21 and 14-3-3? Promoters In Vivo and Has Transcriptional Repressor Activity That Is Reduced by Hay-Wells Syndrome-Derived Mutations

    doi: 10.1128/MCB.23.7.2264-2276.2003

    Figure Lengend Snippet: Relative binding affinities of p53 and ΔNp63α for p53 consensus sites in the p21 and 14-3-3σ promoters. (A) The p53 consensus binding sequence and p53 binding sites in the p21 and 14-3-3σ promoters. Abbreviations: R, purine; Y, pyrimidine; W, adenine or thymine. (B) H1299 cells were transfected with myc-tagged p53 or ΔNp63α, and protein lysates were quantified by using the Fluor-S Max MultiImager. Based on Fluor-S Max quantification, equal amounts of myc-tagged p53 and myc-tagged ΔNp63α were immunoprecipitated with a myc epitope antibody. Immunoprecipitated p53 and ΔNp63α were assayed for their ability to bind radiolabeled oligonucleotides representing p53-binding sites in the p21 and 14-3-3σ promoters as described in Materials and Methods. (C) Bound oligonucleotides were separated on acrylamide gels, exposed for autoradiography, and quantified. Each autoradiograph shows one representative result of at least three independent experiments that are quantified and displayed with the standard deviation.

    Article Snippet: Western analyses of the transfected cell lysates with α-myc antibody (clone 9E-10) were performed, and p53 and p63 were quantified in triplicate by using a Fluor-S Max MultiImager (Bio-Rad Laboratories).

    Techniques: Binding Assay, Sequencing, Transfection, Immunoprecipitation, Autoradiography, Standard Deviation