tla 55 rotor  (Beckman Coulter)


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  • 96

    Structured Review

    Beckman Coulter tla 55 rotor
    Y14D mutant readily dissociates into monomers in nonboiled samples, shifts the distribution toward monomers on sucrose gradients, and partitions into the cytosolic fraction. (A) Western blot analysis of boiled and nonboiled lysates of Myc-tagged Cav1 constructs in Cav1 − / − MLECs. To detect Cav1 − / − MLECs expressing WT-Cav1, Y14F, Y14D and C156S mutants were lysed and blotted using standard conditions (including RIPA buffer and boiling) or gently solubilized in saline buffer supplemented with 2% ODG and heated at 60°C to preserve higher- order oligomers (“nonboiled”). Membranes were blotted with anti-Cav1 pAb. HEK cells expressing (B) WT-Cav1, (C) Y14F-Cav1, or (D) Y14D-Cav1 were fractionated by density gradient centrifugation on 5–30% sucrose gradients. Eleven equal fractions, with the top fraction (1) being the lightest and bottom fraction (11) the heaviest, were analyzed by Western blot using anti–total Cav1 pAb. Bar graphs depicting the distribution (relative abundance) of Cav1 oligomers and monomers in each lane were calculated as a percentage of the total Cav1 in the entire blot. (E) Analysis of cytosolic and total membrane fractions from Cav1 − / − MLECs expressing Myc-tagged Cav1 cDNAs. Detergent-free cell homogenates were clarified by low-speed spin (500 × g ) to remove nuclei and unbroken cells, and then supernatants were ultracentrifuged at 55,000 rpm in a <t>TLA-55</t> Beckman rotor to separate supernatant (cytosol/microvesicle fraction) from the pelleted total membrane fraction (pellet). The two fractions were then solubilized and adjusted to the same concentration, heated in sample buffer at 60°C (“nonboiled”), and resolved on SDS–PAGE gels, followed by detection with anti–total Cav1 pAb. The C156S Cav1 mutant was used as a negative control for Cav1 oligomerization ( Bakhshi et al ., 2013 ).
    Tla 55 Rotor, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 96/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae"

    Article Title: Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E15-11-0756

    Y14D mutant readily dissociates into monomers in nonboiled samples, shifts the distribution toward monomers on sucrose gradients, and partitions into the cytosolic fraction. (A) Western blot analysis of boiled and nonboiled lysates of Myc-tagged Cav1 constructs in Cav1 − / − MLECs. To detect Cav1 − / − MLECs expressing WT-Cav1, Y14F, Y14D and C156S mutants were lysed and blotted using standard conditions (including RIPA buffer and boiling) or gently solubilized in saline buffer supplemented with 2% ODG and heated at 60°C to preserve higher- order oligomers (“nonboiled”). Membranes were blotted with anti-Cav1 pAb. HEK cells expressing (B) WT-Cav1, (C) Y14F-Cav1, or (D) Y14D-Cav1 were fractionated by density gradient centrifugation on 5–30% sucrose gradients. Eleven equal fractions, with the top fraction (1) being the lightest and bottom fraction (11) the heaviest, were analyzed by Western blot using anti–total Cav1 pAb. Bar graphs depicting the distribution (relative abundance) of Cav1 oligomers and monomers in each lane were calculated as a percentage of the total Cav1 in the entire blot. (E) Analysis of cytosolic and total membrane fractions from Cav1 − / − MLECs expressing Myc-tagged Cav1 cDNAs. Detergent-free cell homogenates were clarified by low-speed spin (500 × g ) to remove nuclei and unbroken cells, and then supernatants were ultracentrifuged at 55,000 rpm in a TLA-55 Beckman rotor to separate supernatant (cytosol/microvesicle fraction) from the pelleted total membrane fraction (pellet). The two fractions were then solubilized and adjusted to the same concentration, heated in sample buffer at 60°C (“nonboiled”), and resolved on SDS–PAGE gels, followed by detection with anti–total Cav1 pAb. The C156S Cav1 mutant was used as a negative control for Cav1 oligomerization ( Bakhshi et al ., 2013 ).
    Figure Legend Snippet: Y14D mutant readily dissociates into monomers in nonboiled samples, shifts the distribution toward monomers on sucrose gradients, and partitions into the cytosolic fraction. (A) Western blot analysis of boiled and nonboiled lysates of Myc-tagged Cav1 constructs in Cav1 − / − MLECs. To detect Cav1 − / − MLECs expressing WT-Cav1, Y14F, Y14D and C156S mutants were lysed and blotted using standard conditions (including RIPA buffer and boiling) or gently solubilized in saline buffer supplemented with 2% ODG and heated at 60°C to preserve higher- order oligomers (“nonboiled”). Membranes were blotted with anti-Cav1 pAb. HEK cells expressing (B) WT-Cav1, (C) Y14F-Cav1, or (D) Y14D-Cav1 were fractionated by density gradient centrifugation on 5–30% sucrose gradients. Eleven equal fractions, with the top fraction (1) being the lightest and bottom fraction (11) the heaviest, were analyzed by Western blot using anti–total Cav1 pAb. Bar graphs depicting the distribution (relative abundance) of Cav1 oligomers and monomers in each lane were calculated as a percentage of the total Cav1 in the entire blot. (E) Analysis of cytosolic and total membrane fractions from Cav1 − / − MLECs expressing Myc-tagged Cav1 cDNAs. Detergent-free cell homogenates were clarified by low-speed spin (500 × g ) to remove nuclei and unbroken cells, and then supernatants were ultracentrifuged at 55,000 rpm in a TLA-55 Beckman rotor to separate supernatant (cytosol/microvesicle fraction) from the pelleted total membrane fraction (pellet). The two fractions were then solubilized and adjusted to the same concentration, heated in sample buffer at 60°C (“nonboiled”), and resolved on SDS–PAGE gels, followed by detection with anti–total Cav1 pAb. The C156S Cav1 mutant was used as a negative control for Cav1 oligomerization ( Bakhshi et al ., 2013 ).

    Techniques Used: Mutagenesis, Western Blot, Construct, Expressing, Gradient Centrifugation, Concentration Assay, SDS Page, Negative Control

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    Beckman Coulter cscl centrifugation
    SIPSim output provides data that approximates results obtained from <t>DNA-SIP</t> experiments. The <t>CsCl</t> gradient BD distributions of diverse amplicon fragments ( n = 1,147 taxa) are depicted such that the distribution of each taxon is represented by a different color. All taxa in the control had 0% atom excess 13 C, while 10% of taxa in the treatment were randomly assigned 100% atom excess 13 C. Most unlabeled amplicon fragments occur within the range of 1.69–1.72 g ml −1 , while 13 C-labeled taxa are shifted into higher BD fractions (pre-sequencing, top panels). During the process of high-throughput DNA sequencing amplicon fragments are randomly sampled from each fraction, and this sampling effect alters the shape of the fragment distributions observed in DNA-SIP experiments (post-sequencing, middle panel) relative to the actual distribution of DNA in the gradient (top panels). Typically, data from DNA-SIP experiments are transformed into relative abundance values (post-sequencing, bottom panel) prior to analysis. Identification of taxa that have incorporated isotope requires comparison of amplicon fragment relative abundance distributions in treatment relative to control gradients. The dashed vertical line is provided as a point of reference and designates the theoretical buoyant density of an unlabeled DNA fragment with 50% G + C (as modeled in Equation 1).
    Cscl Centrifugation, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Beckman Coulter tla 55 rotor
    Y14D mutant readily dissociates into monomers in nonboiled samples, shifts the distribution toward monomers on sucrose gradients, and partitions into the cytosolic fraction. (A) Western blot analysis of boiled and nonboiled lysates of Myc-tagged Cav1 constructs in Cav1 − / − MLECs. To detect Cav1 − / − MLECs expressing WT-Cav1, Y14F, Y14D and C156S mutants were lysed and blotted using standard conditions (including RIPA buffer and boiling) or gently solubilized in saline buffer supplemented with 2% ODG and heated at 60°C to preserve higher- order oligomers (“nonboiled”). Membranes were blotted with anti-Cav1 pAb. HEK cells expressing (B) WT-Cav1, (C) Y14F-Cav1, or (D) Y14D-Cav1 were fractionated by density gradient centrifugation on 5–30% sucrose gradients. Eleven equal fractions, with the top fraction (1) being the lightest and bottom fraction (11) the heaviest, were analyzed by Western blot using anti–total Cav1 pAb. Bar graphs depicting the distribution (relative abundance) of Cav1 oligomers and monomers in each lane were calculated as a percentage of the total Cav1 in the entire blot. (E) Analysis of cytosolic and total membrane fractions from Cav1 − / − MLECs expressing Myc-tagged Cav1 cDNAs. Detergent-free cell homogenates were clarified by low-speed spin (500 × g ) to remove nuclei and unbroken cells, and then supernatants were ultracentrifuged at 55,000 rpm in a <t>TLA-55</t> Beckman rotor to separate supernatant (cytosol/microvesicle fraction) from the pelleted total membrane fraction (pellet). The two fractions were then solubilized and adjusted to the same concentration, heated in sample buffer at 60°C (“nonboiled”), and resolved on SDS–PAGE gels, followed by detection with anti–total Cav1 pAb. The C156S Cav1 mutant was used as a negative control for Cav1 oligomerization ( Bakhshi et al ., 2013 ).
    Tla 55 Rotor, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tla 55 rotor/product/Beckman Coulter
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tla 55 rotor - by Bioz Stars, 2022-10
    96/100 stars
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    Image Search Results


    SIPSim output provides data that approximates results obtained from DNA-SIP experiments. The CsCl gradient BD distributions of diverse amplicon fragments ( n = 1,147 taxa) are depicted such that the distribution of each taxon is represented by a different color. All taxa in the control had 0% atom excess 13 C, while 10% of taxa in the treatment were randomly assigned 100% atom excess 13 C. Most unlabeled amplicon fragments occur within the range of 1.69–1.72 g ml −1 , while 13 C-labeled taxa are shifted into higher BD fractions (pre-sequencing, top panels). During the process of high-throughput DNA sequencing amplicon fragments are randomly sampled from each fraction, and this sampling effect alters the shape of the fragment distributions observed in DNA-SIP experiments (post-sequencing, middle panel) relative to the actual distribution of DNA in the gradient (top panels). Typically, data from DNA-SIP experiments are transformed into relative abundance values (post-sequencing, bottom panel) prior to analysis. Identification of taxa that have incorporated isotope requires comparison of amplicon fragment relative abundance distributions in treatment relative to control gradients. The dashed vertical line is provided as a point of reference and designates the theoretical buoyant density of an unlabeled DNA fragment with 50% G + C (as modeled in Equation 1).

    Journal: Frontiers in Microbiology

    Article Title: SIPSim: A Modeling Toolkit to Predict Accuracy and Aid Design of DNA-SIP Experiments

    doi: 10.3389/fmicb.2018.00570

    Figure Lengend Snippet: SIPSim output provides data that approximates results obtained from DNA-SIP experiments. The CsCl gradient BD distributions of diverse amplicon fragments ( n = 1,147 taxa) are depicted such that the distribution of each taxon is represented by a different color. All taxa in the control had 0% atom excess 13 C, while 10% of taxa in the treatment were randomly assigned 100% atom excess 13 C. Most unlabeled amplicon fragments occur within the range of 1.69–1.72 g ml −1 , while 13 C-labeled taxa are shifted into higher BD fractions (pre-sequencing, top panels). During the process of high-throughput DNA sequencing amplicon fragments are randomly sampled from each fraction, and this sampling effect alters the shape of the fragment distributions observed in DNA-SIP experiments (post-sequencing, middle panel) relative to the actual distribution of DNA in the gradient (top panels). Typically, data from DNA-SIP experiments are transformed into relative abundance values (post-sequencing, bottom panel) prior to analysis. Identification of taxa that have incorporated isotope requires comparison of amplicon fragment relative abundance distributions in treatment relative to control gradients. The dashed vertical line is provided as a point of reference and designates the theoretical buoyant density of an unlabeled DNA fragment with 50% G + C (as modeled in Equation 1).

    Article Snippet: DNA was subject to CsCl centrifugation (TLA110 Beckman rotor, 55,000 rpm, 66 h, 1.69 g ml−1 average gradient density) and fractionated (100 μl fractions) using methods which have previously been described in detail (Pepe-Ranney et al., ).

    Techniques: Amplification, Labeling, Sequencing, High Throughput Screening Assay, DNA Sequencing, Sampling, Transformation Assay

    Empirical DNA-SIP data shows that unlabeled DNA is found widely within the gradient and that changes in beta-diversity can alter the composition of “heavy” fractions in the absence of isotopically labeled substrates. The DNA is from soil communities incubated for 1, 3, 6, 14, 30, or 48 days following the addition of an unlabeled nutrient mixture. SSU rRNA genes were amplified and sequenced from approximately 24 fractions from each gradient, these amplicons were used to identify the BD variance of amplicon fragments derived from discrete OTUs. The DNA concentration of each gradient fraction was measured using Picogreen assay (A) . These values are normalized to the maximum concentration within each gradient. The amplicon diversity within each gradient fraction was measured using the Shannon Index, showing that the diversity of heavy fractions differs between samples even in the absence of isotopic labeling (B) . The correlograms (C) reveal autocorrelation (measured with Mantel tests) between taxonomic similarity and fraction BD within each gradient. The variance in OTU BD is positively correlated with OTU pre-fractionation relative abundance, with highly abundant OTUs found throughout the CsCl gradient (D) . To improve clarity, single OTUs in (D) were binned into hexagons, with darker shading indicating more OTUs.

    Journal: Frontiers in Microbiology

    Article Title: SIPSim: A Modeling Toolkit to Predict Accuracy and Aid Design of DNA-SIP Experiments

    doi: 10.3389/fmicb.2018.00570

    Figure Lengend Snippet: Empirical DNA-SIP data shows that unlabeled DNA is found widely within the gradient and that changes in beta-diversity can alter the composition of “heavy” fractions in the absence of isotopically labeled substrates. The DNA is from soil communities incubated for 1, 3, 6, 14, 30, or 48 days following the addition of an unlabeled nutrient mixture. SSU rRNA genes were amplified and sequenced from approximately 24 fractions from each gradient, these amplicons were used to identify the BD variance of amplicon fragments derived from discrete OTUs. The DNA concentration of each gradient fraction was measured using Picogreen assay (A) . These values are normalized to the maximum concentration within each gradient. The amplicon diversity within each gradient fraction was measured using the Shannon Index, showing that the diversity of heavy fractions differs between samples even in the absence of isotopic labeling (B) . The correlograms (C) reveal autocorrelation (measured with Mantel tests) between taxonomic similarity and fraction BD within each gradient. The variance in OTU BD is positively correlated with OTU pre-fractionation relative abundance, with highly abundant OTUs found throughout the CsCl gradient (D) . To improve clarity, single OTUs in (D) were binned into hexagons, with darker shading indicating more OTUs.

    Article Snippet: DNA was subject to CsCl centrifugation (TLA110 Beckman rotor, 55,000 rpm, 66 h, 1.69 g ml−1 average gradient density) and fractionated (100 μl fractions) using methods which have previously been described in detail (Pepe-Ranney et al., ).

    Techniques: Labeling, Incubation, Amplification, Derivative Assay, Concentration Assay, Picogreen Assay, Isotopic Labeling, Fractionation

    SIPSim output provides data that approximates results obtained from DNA-SIP experiments. The CsCl gradient BD distributions of diverse amplicon fragments ( n = 1,147 taxa) are depicted such that the distribution of each taxon is represented by a different color. All taxa in the control had 0% atom excess 13 C, while 10% of taxa in the treatment were randomly assigned 100% atom excess 13 C. Most unlabeled amplicon fragments occur within the range of 1.69–1.72 g ml −1 , while 13 C-labeled taxa are shifted into higher BD fractions (pre-sequencing, top panels). During the process of high-throughput DNA sequencing amplicon fragments are randomly sampled from each fraction, and this sampling effect alters the shape of the fragment distributions observed in DNA-SIP experiments (post-sequencing, middle panel) relative to the actual distribution of DNA in the gradient (top panels). Typically, data from DNA-SIP experiments are transformed into relative abundance values (post-sequencing, bottom panel) prior to analysis. Identification of taxa that have incorporated isotope requires comparison of amplicon fragment relative abundance distributions in treatment relative to control gradients. The dashed vertical line is provided as a point of reference and designates the theoretical buoyant density of an unlabeled DNA fragment with 50% G + C (as modeled in Equation 1).

    Journal: Frontiers in Microbiology

    Article Title: SIPSim: A Modeling Toolkit to Predict Accuracy and Aid Design of DNA-SIP Experiments

    doi: 10.3389/fmicb.2018.00570

    Figure Lengend Snippet: SIPSim output provides data that approximates results obtained from DNA-SIP experiments. The CsCl gradient BD distributions of diverse amplicon fragments ( n = 1,147 taxa) are depicted such that the distribution of each taxon is represented by a different color. All taxa in the control had 0% atom excess 13 C, while 10% of taxa in the treatment were randomly assigned 100% atom excess 13 C. Most unlabeled amplicon fragments occur within the range of 1.69–1.72 g ml −1 , while 13 C-labeled taxa are shifted into higher BD fractions (pre-sequencing, top panels). During the process of high-throughput DNA sequencing amplicon fragments are randomly sampled from each fraction, and this sampling effect alters the shape of the fragment distributions observed in DNA-SIP experiments (post-sequencing, middle panel) relative to the actual distribution of DNA in the gradient (top panels). Typically, data from DNA-SIP experiments are transformed into relative abundance values (post-sequencing, bottom panel) prior to analysis. Identification of taxa that have incorporated isotope requires comparison of amplicon fragment relative abundance distributions in treatment relative to control gradients. The dashed vertical line is provided as a point of reference and designates the theoretical buoyant density of an unlabeled DNA fragment with 50% G + C (as modeled in Equation 1).

    Article Snippet: DNA was subject to CsCl centrifugation (TLA110 Beckman rotor, 55,000 rpm, 66 h, 1.69 g ml−1 .

    Techniques: Amplification, Labeling, Sequencing, High Throughput Screening Assay, DNA Sequencing, Sampling, Transformation Assay

    Empirical DNA-SIP data shows that unlabeled DNA is found widely within the gradient and that changes in beta-diversity can alter the composition of “heavy” fractions in the absence of isotopically labeled substrates. The DNA is from soil communities incubated for 1, 3, 6, 14, 30, or 48 days following the addition of an unlabeled nutrient mixture. SSU rRNA genes were amplified and sequenced from approximately 24 fractions from each gradient, these amplicons were used to identify the BD variance of amplicon fragments derived from discrete OTUs. The DNA concentration of each gradient fraction was measured using Picogreen assay (A) . These values are normalized to the maximum concentration within each gradient. The amplicon diversity within each gradient fraction was measured using the Shannon Index, showing that the diversity of heavy fractions differs between samples even in the absence of isotopic labeling (B) . The correlograms (C) reveal autocorrelation (measured with Mantel tests) between taxonomic similarity and fraction BD within each gradient. The variance in OTU BD is positively correlated with OTU pre-fractionation relative abundance, with highly abundant OTUs found throughout the CsCl gradient (D) . To improve clarity, single OTUs in (D) were binned into hexagons, with darker shading indicating more OTUs.

    Journal: Frontiers in Microbiology

    Article Title: SIPSim: A Modeling Toolkit to Predict Accuracy and Aid Design of DNA-SIP Experiments

    doi: 10.3389/fmicb.2018.00570

    Figure Lengend Snippet: Empirical DNA-SIP data shows that unlabeled DNA is found widely within the gradient and that changes in beta-diversity can alter the composition of “heavy” fractions in the absence of isotopically labeled substrates. The DNA is from soil communities incubated for 1, 3, 6, 14, 30, or 48 days following the addition of an unlabeled nutrient mixture. SSU rRNA genes were amplified and sequenced from approximately 24 fractions from each gradient, these amplicons were used to identify the BD variance of amplicon fragments derived from discrete OTUs. The DNA concentration of each gradient fraction was measured using Picogreen assay (A) . These values are normalized to the maximum concentration within each gradient. The amplicon diversity within each gradient fraction was measured using the Shannon Index, showing that the diversity of heavy fractions differs between samples even in the absence of isotopic labeling (B) . The correlograms (C) reveal autocorrelation (measured with Mantel tests) between taxonomic similarity and fraction BD within each gradient. The variance in OTU BD is positively correlated with OTU pre-fractionation relative abundance, with highly abundant OTUs found throughout the CsCl gradient (D) . To improve clarity, single OTUs in (D) were binned into hexagons, with darker shading indicating more OTUs.

    Article Snippet: DNA was subject to CsCl centrifugation (TLA110 Beckman rotor, 55,000 rpm, 66 h, 1.69 g ml−1 .

    Techniques: Labeling, Incubation, Amplification, Derivative Assay, Concentration Assay, Picogreen Assay, Isotopic Labeling, Fractionation

    Y14D mutant readily dissociates into monomers in nonboiled samples, shifts the distribution toward monomers on sucrose gradients, and partitions into the cytosolic fraction. (A) Western blot analysis of boiled and nonboiled lysates of Myc-tagged Cav1 constructs in Cav1 − / − MLECs. To detect Cav1 − / − MLECs expressing WT-Cav1, Y14F, Y14D and C156S mutants were lysed and blotted using standard conditions (including RIPA buffer and boiling) or gently solubilized in saline buffer supplemented with 2% ODG and heated at 60°C to preserve higher- order oligomers (“nonboiled”). Membranes were blotted with anti-Cav1 pAb. HEK cells expressing (B) WT-Cav1, (C) Y14F-Cav1, or (D) Y14D-Cav1 were fractionated by density gradient centrifugation on 5–30% sucrose gradients. Eleven equal fractions, with the top fraction (1) being the lightest and bottom fraction (11) the heaviest, were analyzed by Western blot using anti–total Cav1 pAb. Bar graphs depicting the distribution (relative abundance) of Cav1 oligomers and monomers in each lane were calculated as a percentage of the total Cav1 in the entire blot. (E) Analysis of cytosolic and total membrane fractions from Cav1 − / − MLECs expressing Myc-tagged Cav1 cDNAs. Detergent-free cell homogenates were clarified by low-speed spin (500 × g ) to remove nuclei and unbroken cells, and then supernatants were ultracentrifuged at 55,000 rpm in a TLA-55 Beckman rotor to separate supernatant (cytosol/microvesicle fraction) from the pelleted total membrane fraction (pellet). The two fractions were then solubilized and adjusted to the same concentration, heated in sample buffer at 60°C (“nonboiled”), and resolved on SDS–PAGE gels, followed by detection with anti–total Cav1 pAb. The C156S Cav1 mutant was used as a negative control for Cav1 oligomerization ( Bakhshi et al ., 2013 ).

    Journal: Molecular Biology of the Cell

    Article Title: Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae

    doi: 10.1091/mbc.E15-11-0756

    Figure Lengend Snippet: Y14D mutant readily dissociates into monomers in nonboiled samples, shifts the distribution toward monomers on sucrose gradients, and partitions into the cytosolic fraction. (A) Western blot analysis of boiled and nonboiled lysates of Myc-tagged Cav1 constructs in Cav1 − / − MLECs. To detect Cav1 − / − MLECs expressing WT-Cav1, Y14F, Y14D and C156S mutants were lysed and blotted using standard conditions (including RIPA buffer and boiling) or gently solubilized in saline buffer supplemented with 2% ODG and heated at 60°C to preserve higher- order oligomers (“nonboiled”). Membranes were blotted with anti-Cav1 pAb. HEK cells expressing (B) WT-Cav1, (C) Y14F-Cav1, or (D) Y14D-Cav1 were fractionated by density gradient centrifugation on 5–30% sucrose gradients. Eleven equal fractions, with the top fraction (1) being the lightest and bottom fraction (11) the heaviest, were analyzed by Western blot using anti–total Cav1 pAb. Bar graphs depicting the distribution (relative abundance) of Cav1 oligomers and monomers in each lane were calculated as a percentage of the total Cav1 in the entire blot. (E) Analysis of cytosolic and total membrane fractions from Cav1 − / − MLECs expressing Myc-tagged Cav1 cDNAs. Detergent-free cell homogenates were clarified by low-speed spin (500 × g ) to remove nuclei and unbroken cells, and then supernatants were ultracentrifuged at 55,000 rpm in a TLA-55 Beckman rotor to separate supernatant (cytosol/microvesicle fraction) from the pelleted total membrane fraction (pellet). The two fractions were then solubilized and adjusted to the same concentration, heated in sample buffer at 60°C (“nonboiled”), and resolved on SDS–PAGE gels, followed by detection with anti–total Cav1 pAb. The C156S Cav1 mutant was used as a negative control for Cav1 oligomerization ( Bakhshi et al ., 2013 ).

    Article Snippet: The clarified supernatant was spun in a TLA-55 rotor (Beckman) for 30 min at 50,000 rpm.

    Techniques: Mutagenesis, Western Blot, Construct, Expressing, Gradient Centrifugation, Concentration Assay, SDS Page, Negative Control