igepal ca-630 Search Results


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  • 99
    Thermo Fisher triton x 114
    PI-PLC treatment and phase separation of α 2 δ-1, α 2 δ-2 and PrP in mouse cerebella ( A ) DRM fractions from WT (left-hand panel), PrP Tg(WT) (middle panel) and PrP KO (right-hand panel) mouse cerebella were prepared as described in the Materials and methods section. Aliquots were resolved on 3–8% Tris/acetate (to resolve α 2 δs) or 4–12% Bis-Tris gels (to resolve PrP in the same samples), and analysed by Western blotting (WB) with relevant antibodies as indicated. The full profile is not shown, but only the fractions of the sucrose gradient corresponding to DRMs identified by the presence of flotillin-1 (fractions 4–7 harvested from the top). The anti-α 2 δ-1 and anti-α 2 δ-2 antibodies recognize the α 2 -1 and α 2 -2 moieties. ( B ) Aliquots of concentrated DRM fractions from WT (left-hand panel), PrP Tg(WT) (middle panel) and PrP KO (right-hand panel) cerebella were treated with PNGase F and analysed by Western blotting with the indicated antibodies. ( C ) DRM fractions analysed in ( A ) were subjected to PI-PLC treatment and Triton X-114 phase separation (see the Materials and methods section), followed by PNGase F deglycosylation. The proteins remaining in the aqueous (aq) and detergent (det) phase were then resolved on 4–12% Bis-Tris gels and analysed with the indicated antibodies. Lanes 1 and 2 in each panel are from detergent phase fractions, whereas lanes 3 and 4 are from the respective aqueous phase, treated or not with PI-PLC as indicated. ( D ) The PrP Tg(WT) fractions from ( C , middle panel) were also blotted for PrP using the 3F4 antibody. Molecular mass is shown on the right-hand side of the gels in kDa.
    Triton X 114, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore igepal ca630
    (A) Dynamic Light Scattering analysis of E2-T1 . E2-T1 protein was dialysed into 50 mM Tris (pH 7.0) containing 0.2% <t>Igepal</t> <t>CA630.</t> The graph shows size distribution by intensity for physical size determination. Six measurements of 30 readings each were performed. (B) Western blot of purified E2-T1. E2-T1 protein was run under non-reducing conditions and probed with mouse sera raised against E2-T1.
    Igepal Ca630, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 881 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore np 40 igepal
    (A) Dynamic Light Scattering analysis of E2-T1 . E2-T1 protein was dialysed into 50 mM Tris (pH 7.0) containing 0.2% <t>Igepal</t> <t>CA630.</t> The graph shows size distribution by intensity for physical size determination. Six measurements of 30 readings each were performed. (B) Western blot of purified E2-T1. E2-T1 protein was run under non-reducing conditions and probed with mouse sera raised against E2-T1.
    Np 40 Igepal, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore np 40 buffer
    Ubiquitination of ATG14L by ZBTB16 and Cullin3. ( A ) 293T cells were transfected with expression vectors of FLAG-ZBTB16 and Myc-Atg14L and cultured for 24 hr. The cells were treated with MG132 (10 µM) for the last 4 hr before harvesting and then lysed in <t>NP-40</t> buffer. The lysates were immunoprecipitated with anti-ATG14L antibody, and the immunocomplexes were analyzed by western blotting using anti-Flag antibody. The data are expressed as the mean of 3 biological replicates (mean ± SD). Statistical significance was determined by a two-tailed, unpaired Student's t-test. p
    Np 40 Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 725 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore non idet p 40 np40
    Ubiquitination of ATG14L by ZBTB16 and Cullin3. ( A ) 293T cells were transfected with expression vectors of FLAG-ZBTB16 and Myc-Atg14L and cultured for 24 hr. The cells were treated with MG132 (10 µM) for the last 4 hr before harvesting and then lysed in <t>NP-40</t> buffer. The lysates were immunoprecipitated with anti-ATG14L antibody, and the immunocomplexes were analyzed by western blotting using anti-Flag antibody. The data are expressed as the mean of 3 biological replicates (mean ± SD). Statistical significance was determined by a two-tailed, unpaired Student's t-test. p
    Non Idet P 40 Np40, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore np 40 detergent
    Antibodies to GW182-immunoprecipitated Ago2, Dicer and XRN1 proteins from HeLa cell extracts as examined by WB analysis. GW182 and associated proteins were immunoprecipitated using human sera 18033 containing antibodies to GW182 coupled with Protein A sepharose beads and by mouse anti-GW182 monoclonal 4B6 coupled to Protein G sepharose beads. Normal human serum (NHS), IC6 human serum and mouse monoclonal antibody to golgin 97 were used as IP controls. Immunopreciptates were carried out with buffer that did not contain detergent. Proteins (40 μg) were resolved on a 10% SDS-PAGE gel and were detected with a 4B6 monoclonal antibody ( a ) or 18033 human anti-GWB serum ( b ). Immunopreciptates were carried out with buffer that contained 0.3% <t>NP-40</t> detergent. Proteins (40 μg) were resolved on a 6.5% SDS-PAGE gel and were detected using a mouse anti-Ago2 monoclonal 4F9 antibody ( c ). Protein (40 μg) from whole cell extracts or 18033 immunoprecipitates carried out using 0.3% NP-40 buffer were separated on a 6.5% SDS-PAGE gel and probed with NHS or antibodies to golgin 97, GW182, Ago2, XRN1 or Dicer. ( d .
    Np 40 Detergent, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore np 40 solution
    Antibodies to GW182-immunoprecipitated Ago2, Dicer and XRN1 proteins from HeLa cell extracts as examined by WB analysis. GW182 and associated proteins were immunoprecipitated using human sera 18033 containing antibodies to GW182 coupled with Protein A sepharose beads and by mouse anti-GW182 monoclonal 4B6 coupled to Protein G sepharose beads. Normal human serum (NHS), IC6 human serum and mouse monoclonal antibody to golgin 97 were used as IP controls. Immunopreciptates were carried out with buffer that did not contain detergent. Proteins (40 μg) were resolved on a 10% SDS-PAGE gel and were detected with a 4B6 monoclonal antibody ( a ) or 18033 human anti-GWB serum ( b ). Immunopreciptates were carried out with buffer that contained 0.3% <t>NP-40</t> detergent. Proteins (40 μg) were resolved on a 6.5% SDS-PAGE gel and were detected using a mouse anti-Ago2 monoclonal 4F9 antibody ( c ). Protein (40 μg) from whole cell extracts or 18033 immunoprecipitates carried out using 0.3% NP-40 buffer were separated on a 6.5% SDS-PAGE gel and probed with NHS or antibodies to golgin 97, GW182, Ago2, XRN1 or Dicer. ( d .
    Np 40 Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore triton x 114 extraction buffer
    Sialylated mucins are included in lipid rafts whereas TS is not. (A) Cold Triton X-100 partition. Sialylated trypomastigotes were lysed at 4°C. Mucins were predominantly recovered in the pellet whereas TS and HSP70, a cytosolic protein, were recovered in the supernatant. (B) Triton X-114 extraction for GPI-anchored proteins. Parasites were lysed at 4°C and detergent and aqueous phases separated at 37°C and analyzed by Western blots. Mucins and TS partitioned in the detergent phase due to their GPI-anchoring. Glutamate Dehydrogenase, a cytosolic protein, was recovered in the aqueous phase. (C) Purification of DRMs by step-gradient ultracentrifugation. Trypomastigotes were lysed in Triton X-100 at 4°C or 37°C and centrifuged in an Optiprep gradient. Mucins floated to the 35%-5% interface (lane 6) only when lysis was done at 4°C indicating its DRM nature in contrast to TS. (D) Living parasites were sialylated from a Neu5Az donor, then treated for membrane fluidization with 1% diethyl ether in phosphate-buffered saline (PBS) and fixed with  p -formaldehyde (PFA). Doted labeling for TS and mucins was disrupted only after 90sec treatment even showing colocalization.
    Triton X 114 Extraction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore np 40 pbs
    Sialylated mucins are included in lipid rafts whereas TS is not. (A) Cold Triton X-100 partition. Sialylated trypomastigotes were lysed at 4°C. Mucins were predominantly recovered in the pellet whereas TS and HSP70, a cytosolic protein, were recovered in the supernatant. (B) Triton X-114 extraction for GPI-anchored proteins. Parasites were lysed at 4°C and detergent and aqueous phases separated at 37°C and analyzed by Western blots. Mucins and TS partitioned in the detergent phase due to their GPI-anchoring. Glutamate Dehydrogenase, a cytosolic protein, was recovered in the aqueous phase. (C) Purification of DRMs by step-gradient ultracentrifugation. Trypomastigotes were lysed in Triton X-100 at 4°C or 37°C and centrifuged in an Optiprep gradient. Mucins floated to the 35%-5% interface (lane 6) only when lysis was done at 4°C indicating its DRM nature in contrast to TS. (D) Living parasites were sialylated from a Neu5Az donor, then treated for membrane fluidization with 1% diethyl ether in phosphate-buffered saline (PBS) and fixed with  p -formaldehyde (PFA). Doted labeling for TS and mucins was disrupted only after 90sec treatment even showing colocalization.
    Np 40 Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore np 40 lysis buffer
    Binding assays of deuterium-substituted ( S )- and ( R )-thalidomides with human CRBN TBD. ( a ) Chemical structures of deuterated ( S )- and ( R )-thalidomides, ( S )-D-Thal and ( R )-D-Thal, respectively. Atom numbering is shown in the ( S )-D-Thal chemical structure. The hydrogen atom at the chiral centre C3 atom of the glutarimide moiety is substituted with a deuterium atom. ( b ) Competitive elution assay using thalidomide-immobilized beads coupled with racemic thalidomide. Beads were mixed with extracts from 293 T cells expressing FLAG-HA-CRBN and washed three times with 0.5% <t>NP-40</t> lysis buffer and bound proteins were eluted with wash buffer containing 1 mM deuterated ( S )- or ( R )-thalidomide (( S )-D-Thal or ( R )-D-Thal), ( S )- or ( R )-thalidomide (( S )-Thal or ( R )-Thal) or DMSO for the indicated time. The eluate was then analyzed by SDS-PAGE and immunoblotting (IB). ( c ) Inhibitory effects of thalidomide enantiomers on auto-ubiquitylation of FH-CRBN were detected in the presence of MG132. Cells were treated with DMSO or the indicated concentrations of ( S )-D-Thal or ( S )-D-Thal for 4 hours prior to harvesting. Full-length blots in ( b ) and ( c ) are presented in Supplementary Fig. 8 .
    Np 40 Lysis Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2370 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore wash buffer
    Binding assays of deuterium-substituted ( S )- and ( R )-thalidomides with human CRBN TBD. ( a ) Chemical structures of deuterated ( S )- and ( R )-thalidomides, ( S )-D-Thal and ( R )-D-Thal, respectively. Atom numbering is shown in the ( S )-D-Thal chemical structure. The hydrogen atom at the chiral centre C3 atom of the glutarimide moiety is substituted with a deuterium atom. ( b ) Competitive elution assay using thalidomide-immobilized beads coupled with racemic thalidomide. Beads were mixed with extracts from 293 T cells expressing FLAG-HA-CRBN and washed three times with 0.5% <t>NP-40</t> lysis buffer and bound proteins were eluted with wash buffer containing 1 mM deuterated ( S )- or ( R )-thalidomide (( S )-D-Thal or ( R )-D-Thal), ( S )- or ( R )-thalidomide (( S )-Thal or ( R )-Thal) or DMSO for the indicated time. The eluate was then analyzed by SDS-PAGE and immunoblotting (IB). ( c ) Inhibitory effects of thalidomide enantiomers on auto-ubiquitylation of FH-CRBN were detected in the presence of MG132. Cells were treated with DMSO or the indicated concentrations of ( S )-D-Thal or ( S )-D-Thal for 4 hours prior to harvesting. Full-length blots in ( b ) and ( c ) are presented in Supplementary Fig. 8 .
    Wash Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6545 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    np 40  (Abcam)
    99
    Abcam np 40
    Binding assays of deuterium-substituted ( S )- and ( R )-thalidomides with human CRBN TBD. ( a ) Chemical structures of deuterated ( S )- and ( R )-thalidomides, ( S )-D-Thal and ( R )-D-Thal, respectively. Atom numbering is shown in the ( S )-D-Thal chemical structure. The hydrogen atom at the chiral centre C3 atom of the glutarimide moiety is substituted with a deuterium atom. ( b ) Competitive elution assay using thalidomide-immobilized beads coupled with racemic thalidomide. Beads were mixed with extracts from 293 T cells expressing FLAG-HA-CRBN and washed three times with 0.5% <t>NP-40</t> lysis buffer and bound proteins were eluted with wash buffer containing 1 mM deuterated ( S )- or ( R )-thalidomide (( S )-D-Thal or ( R )-D-Thal), ( S )- or ( R )-thalidomide (( S )-Thal or ( R )-Thal) or DMSO for the indicated time. The eluate was then analyzed by SDS-PAGE and immunoblotting (IB). ( c ) Inhibitory effects of thalidomide enantiomers on auto-ubiquitylation of FH-CRBN were detected in the presence of MG132. Cells were treated with DMSO or the indicated concentrations of ( S )-D-Thal or ( S )-D-Thal for 4 hours prior to harvesting. Full-length blots in ( b ) and ( c ) are presented in Supplementary Fig. 8 .
    Np 40, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Valiant igepal ca 630
    Binding assays of deuterium-substituted ( S )- and ( R )-thalidomides with human CRBN TBD. ( a ) Chemical structures of deuterated ( S )- and ( R )-thalidomides, ( S )-D-Thal and ( R )-D-Thal, respectively. Atom numbering is shown in the ( S )-D-Thal chemical structure. The hydrogen atom at the chiral centre C3 atom of the glutarimide moiety is substituted with a deuterium atom. ( b ) Competitive elution assay using thalidomide-immobilized beads coupled with racemic thalidomide. Beads were mixed with extracts from 293 T cells expressing FLAG-HA-CRBN and washed three times with 0.5% <t>NP-40</t> lysis buffer and bound proteins were eluted with wash buffer containing 1 mM deuterated ( S )- or ( R )-thalidomide (( S )-D-Thal or ( R )-D-Thal), ( S )- or ( R )-thalidomide (( S )-Thal or ( R )-Thal) or DMSO for the indicated time. The eluate was then analyzed by SDS-PAGE and immunoblotting (IB). ( c ) Inhibitory effects of thalidomide enantiomers on auto-ubiquitylation of FH-CRBN were detected in the presence of MG132. Cells were treated with DMSO or the indicated concentrations of ( S )-D-Thal or ( S )-D-Thal for 4 hours prior to harvesting. Full-length blots in ( b ) and ( c ) are presented in Supplementary Fig. 8 .
    Igepal Ca 630, supplied by Valiant, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore extraction buffer
    Binding assays of deuterium-substituted ( S )- and ( R )-thalidomides with human CRBN TBD. ( a ) Chemical structures of deuterated ( S )- and ( R )-thalidomides, ( S )-D-Thal and ( R )-D-Thal, respectively. Atom numbering is shown in the ( S )-D-Thal chemical structure. The hydrogen atom at the chiral centre C3 atom of the glutarimide moiety is substituted with a deuterium atom. ( b ) Competitive elution assay using thalidomide-immobilized beads coupled with racemic thalidomide. Beads were mixed with extracts from 293 T cells expressing FLAG-HA-CRBN and washed three times with 0.5% <t>NP-40</t> lysis buffer and bound proteins were eluted with wash buffer containing 1 mM deuterated ( S )- or ( R )-thalidomide (( S )-D-Thal or ( R )-D-Thal), ( S )- or ( R )-thalidomide (( S )-Thal or ( R )-Thal) or DMSO for the indicated time. The eluate was then analyzed by SDS-PAGE and immunoblotting (IB). ( c ) Inhibitory effects of thalidomide enantiomers on auto-ubiquitylation of FH-CRBN were detected in the presence of MG132. Cells were treated with DMSO or the indicated concentrations of ( S )-D-Thal or ( S )-D-Thal for 4 hours prior to harvesting. Full-length blots in ( b ) and ( c ) are presented in Supplementary Fig. 8 .
    Extraction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6906 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lonza igepal ca 630
    Binding assays of deuterium-substituted ( S )- and ( R )-thalidomides with human CRBN TBD. ( a ) Chemical structures of deuterated ( S )- and ( R )-thalidomides, ( S )-D-Thal and ( R )-D-Thal, respectively. Atom numbering is shown in the ( S )-D-Thal chemical structure. The hydrogen atom at the chiral centre C3 atom of the glutarimide moiety is substituted with a deuterium atom. ( b ) Competitive elution assay using thalidomide-immobilized beads coupled with racemic thalidomide. Beads were mixed with extracts from 293 T cells expressing FLAG-HA-CRBN and washed three times with 0.5% <t>NP-40</t> lysis buffer and bound proteins were eluted with wash buffer containing 1 mM deuterated ( S )- or ( R )-thalidomide (( S )-D-Thal or ( R )-D-Thal), ( S )- or ( R )-thalidomide (( S )-Thal or ( R )-Thal) or DMSO for the indicated time. The eluate was then analyzed by SDS-PAGE and immunoblotting (IB). ( c ) Inhibitory effects of thalidomide enantiomers on auto-ubiquitylation of FH-CRBN were detected in the presence of MG132. Cells were treated with DMSO or the indicated concentrations of ( S )-D-Thal or ( S )-D-Thal for 4 hours prior to harvesting. Full-length blots in ( b ) and ( c ) are presented in Supplementary Fig. 8 .
    Igepal Ca 630, supplied by Lonza, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upstate Biotechnology Inc igepal ca 630
    Binding assays of deuterium-substituted ( S )- and ( R )-thalidomides with human CRBN TBD. ( a ) Chemical structures of deuterated ( S )- and ( R )-thalidomides, ( S )-D-Thal and ( R )-D-Thal, respectively. Atom numbering is shown in the ( S )-D-Thal chemical structure. The hydrogen atom at the chiral centre C3 atom of the glutarimide moiety is substituted with a deuterium atom. ( b ) Competitive elution assay using thalidomide-immobilized beads coupled with racemic thalidomide. Beads were mixed with extracts from 293 T cells expressing FLAG-HA-CRBN and washed three times with 0.5% <t>NP-40</t> lysis buffer and bound proteins were eluted with wash buffer containing 1 mM deuterated ( S )- or ( R )-thalidomide (( S )-D-Thal or ( R )-D-Thal), ( S )- or ( R )-thalidomide (( S )-Thal or ( R )-Thal) or DMSO for the indicated time. The eluate was then analyzed by SDS-PAGE and immunoblotting (IB). ( c ) Inhibitory effects of thalidomide enantiomers on auto-ubiquitylation of FH-CRBN were detected in the presence of MG132. Cells were treated with DMSO or the indicated concentrations of ( S )-D-Thal or ( S )-D-Thal for 4 hours prior to harvesting. Full-length blots in ( b ) and ( c ) are presented in Supplementary Fig. 8 .
    Igepal Ca 630, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck & Co igepal ca 630
    Binding assays of deuterium-substituted ( S )- and ( R )-thalidomides with human CRBN TBD. ( a ) Chemical structures of deuterated ( S )- and ( R )-thalidomides, ( S )-D-Thal and ( R )-D-Thal, respectively. Atom numbering is shown in the ( S )-D-Thal chemical structure. The hydrogen atom at the chiral centre C3 atom of the glutarimide moiety is substituted with a deuterium atom. ( b ) Competitive elution assay using thalidomide-immobilized beads coupled with racemic thalidomide. Beads were mixed with extracts from 293 T cells expressing FLAG-HA-CRBN and washed three times with 0.5% <t>NP-40</t> lysis buffer and bound proteins were eluted with wash buffer containing 1 mM deuterated ( S )- or ( R )-thalidomide (( S )-D-Thal or ( R )-D-Thal), ( S )- or ( R )-thalidomide (( S )-Thal or ( R )-Thal) or DMSO for the indicated time. The eluate was then analyzed by SDS-PAGE and immunoblotting (IB). ( c ) Inhibitory effects of thalidomide enantiomers on auto-ubiquitylation of FH-CRBN were detected in the presence of MG132. Cells were treated with DMSO or the indicated concentrations of ( S )-D-Thal or ( S )-D-Thal for 4 hours prior to harvesting. Full-length blots in ( b ) and ( c ) are presented in Supplementary Fig. 8 .
    Igepal Ca 630, supplied by Merck & Co, used in various techniques. Bioz Stars score: 95/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher np 40 substitute igepal ca 630
    Binding assays of deuterium-substituted ( S )- and ( R )-thalidomides with human CRBN TBD. ( a ) Chemical structures of deuterated ( S )- and ( R )-thalidomides, ( S )-D-Thal and ( R )-D-Thal, respectively. Atom numbering is shown in the ( S )-D-Thal chemical structure. The hydrogen atom at the chiral centre C3 atom of the glutarimide moiety is substituted with a deuterium atom. ( b ) Competitive elution assay using thalidomide-immobilized beads coupled with racemic thalidomide. Beads were mixed with extracts from 293 T cells expressing FLAG-HA-CRBN and washed three times with 0.5% <t>NP-40</t> lysis buffer and bound proteins were eluted with wash buffer containing 1 mM deuterated ( S )- or ( R )-thalidomide (( S )-D-Thal or ( R )-D-Thal), ( S )- or ( R )-thalidomide (( S )-Thal or ( R )-Thal) or DMSO for the indicated time. The eluate was then analyzed by SDS-PAGE and immunoblotting (IB). ( c ) Inhibitory effects of thalidomide enantiomers on auto-ubiquitylation of FH-CRBN were detected in the presence of MG132. Cells were treated with DMSO or the indicated concentrations of ( S )-D-Thal or ( S )-D-Thal for 4 hours prior to harvesting. Full-length blots in ( b ) and ( c ) are presented in Supplementary Fig. 8 .
    Np 40 Substitute Igepal Ca 630, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fisher Scientific triton x 114
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    US Biological Life Sciences igepal ca 630
    Effect of low-pH treatment on the hydrophobicity of gB. Soluble gB or BSA was added to 2% Triton X-114 that had been adjusted to the indicated pH. Samples were incubated for 10 min at 37°C and were centrifuged at 300 ×  g  for 3 min. The aqueous supernatant phase and detergent phase were collected and diluted 20-fold in PBS. s-gB samples were subjected to immunoprecipitation with antibody to gB, followed by SDS-PAGE and immunoblotting for gB. BSA samples were trichloroacetic acid (TCA) precipitated and analyzed by SDS-PAGE and Coomassie blue staining.
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    Effect of low-pH treatment on the hydrophobicity of gB. Soluble gB or BSA was added to 2% Triton X-114 that had been adjusted to the indicated pH. Samples were incubated for 10 min at 37°C and were centrifuged at 300 ×  g  for 3 min. The aqueous supernatant phase and detergent phase were collected and diluted 20-fold in PBS. s-gB samples were subjected to immunoprecipitation with antibody to gB, followed by SDS-PAGE and immunoblotting for gB. BSA samples were trichloroacetic acid (TCA) precipitated and analyzed by SDS-PAGE and Coomassie blue staining.
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    Effect of low-pH treatment on the hydrophobicity of gB. Soluble gB or BSA was added to 2% Triton X-114 that had been adjusted to the indicated pH. Samples were incubated for 10 min at 37°C and were centrifuged at 300 ×  g  for 3 min. The aqueous supernatant phase and detergent phase were collected and diluted 20-fold in PBS. s-gB samples were subjected to immunoprecipitation with antibody to gB, followed by SDS-PAGE and immunoblotting for gB. BSA samples were trichloroacetic acid (TCA) precipitated and analyzed by SDS-PAGE and Coomassie blue staining.
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    Effect of low-pH treatment on the hydrophobicity of gB. Soluble gB or BSA was added to 2% Triton X-114 that had been adjusted to the indicated pH. Samples were incubated for 10 min at 37°C and were centrifuged at 300 ×  g  for 3 min. The aqueous supernatant phase and detergent phase were collected and diluted 20-fold in PBS. s-gB samples were subjected to immunoprecipitation with antibody to gB, followed by SDS-PAGE and immunoblotting for gB. BSA samples were trichloroacetic acid (TCA) precipitated and analyzed by SDS-PAGE and Coomassie blue staining.
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    Effect of low-pH treatment on the hydrophobicity of gB. Soluble gB or BSA was added to 2% Triton X-114 that had been adjusted to the indicated pH. Samples were incubated for 10 min at 37°C and were centrifuged at 300 ×  g  for 3 min. The aqueous supernatant phase and detergent phase were collected and diluted 20-fold in PBS. s-gB samples were subjected to immunoprecipitation with antibody to gB, followed by SDS-PAGE and immunoblotting for gB. BSA samples were trichloroacetic acid (TCA) precipitated and analyzed by SDS-PAGE and Coomassie blue staining.
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    Effect of low-pH treatment on the hydrophobicity of gB. Soluble gB or BSA was added to 2% Triton X-114 that had been adjusted to the indicated pH. Samples were incubated for 10 min at 37°C and were centrifuged at 300 ×  g  for 3 min. The aqueous supernatant phase and detergent phase were collected and diluted 20-fold in PBS. s-gB samples were subjected to immunoprecipitation with antibody to gB, followed by SDS-PAGE and immunoblotting for gB. BSA samples were trichloroacetic acid (TCA) precipitated and analyzed by SDS-PAGE and Coomassie blue staining.
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    Effect of low-pH treatment on the hydrophobicity of gB. Soluble gB or BSA was added to 2% Triton X-114 that had been adjusted to the indicated pH. Samples were incubated for 10 min at 37°C and were centrifuged at 300 ×  g  for 3 min. The aqueous supernatant phase and detergent phase were collected and diluted 20-fold in PBS. s-gB samples were subjected to immunoprecipitation with antibody to gB, followed by SDS-PAGE and immunoblotting for gB. BSA samples were trichloroacetic acid (TCA) precipitated and analyzed by SDS-PAGE and Coomassie blue staining.
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    TX-114 extraction demonstrates hydrophobic modifications to Rab lipid tail  variants. Triton X-114 fractionation generating a detergent-enriched and  aqueous phase was performed as described under MATERIALS AND METHODS on cells  expressing GFP-Ypt1p, -Ypt1p CTIM , -Ypt1p CIIL ,  -Ypt1p C205S , -Ypt1p Δ CC , -Sec4p,  -Sec4p CTIM , -Sec4p CIIL , -Sec4p C214S , and  -Sec4p Δ CC . The detergent-enriched phase was then  subjected to trichoroacetic acid precipitation followed by SDS-PAGE  electrophoresis and Western blotting to detect the GFP-fusion proteins. As a  control, the fractions were probed for the transmembrane protein Snc1/2p.  Relevant protein markers are indicated.
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    TX-114 extraction demonstrates hydrophobic modifications to Rab lipid tail  variants. Triton X-114 fractionation generating a detergent-enriched and  aqueous phase was performed as described under MATERIALS AND METHODS on cells  expressing GFP-Ypt1p, -Ypt1p CTIM , -Ypt1p CIIL ,  -Ypt1p C205S , -Ypt1p Δ CC , -Sec4p,  -Sec4p CTIM , -Sec4p CIIL , -Sec4p C214S , and  -Sec4p Δ CC . The detergent-enriched phase was then  subjected to trichoroacetic acid precipitation followed by SDS-PAGE  electrophoresis and Western blotting to detect the GFP-fusion proteins. As a  control, the fractions were probed for the transmembrane protein Snc1/2p.  Relevant protein markers are indicated.
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    TX-114 extraction demonstrates hydrophobic modifications to Rab lipid tail  variants. Triton X-114 fractionation generating a detergent-enriched and  aqueous phase was performed as described under MATERIALS AND METHODS on cells  expressing GFP-Ypt1p, -Ypt1p CTIM , -Ypt1p CIIL ,  -Ypt1p C205S , -Ypt1p Δ CC , -Sec4p,  -Sec4p CTIM , -Sec4p CIIL , -Sec4p C214S , and  -Sec4p Δ CC . The detergent-enriched phase was then  subjected to trichoroacetic acid precipitation followed by SDS-PAGE  electrophoresis and Western blotting to detect the GFP-fusion proteins. As a  control, the fractions were probed for the transmembrane protein Snc1/2p.  Relevant protein markers are indicated.
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    TX-114 extraction demonstrates hydrophobic modifications to Rab lipid tail  variants. Triton X-114 fractionation generating a detergent-enriched and  aqueous phase was performed as described under MATERIALS AND METHODS on cells  expressing GFP-Ypt1p, -Ypt1p CTIM , -Ypt1p CIIL ,  -Ypt1p C205S , -Ypt1p Δ CC , -Sec4p,  -Sec4p CTIM , -Sec4p CIIL , -Sec4p C214S , and  -Sec4p Δ CC . The detergent-enriched phase was then  subjected to trichoroacetic acid precipitation followed by SDS-PAGE  electrophoresis and Western blotting to detect the GFP-fusion proteins. As a  control, the fractions were probed for the transmembrane protein Snc1/2p.  Relevant protein markers are indicated.
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    Image Search Results


    PI-PLC treatment and phase separation of α 2 δ-1, α 2 δ-2 and PrP in mouse cerebella ( A ) DRM fractions from WT (left-hand panel), PrP Tg(WT) (middle panel) and PrP KO (right-hand panel) mouse cerebella were prepared as described in the Materials and methods section. Aliquots were resolved on 3–8% Tris/acetate (to resolve α 2 δs) or 4–12% Bis-Tris gels (to resolve PrP in the same samples), and analysed by Western blotting (WB) with relevant antibodies as indicated. The full profile is not shown, but only the fractions of the sucrose gradient corresponding to DRMs identified by the presence of flotillin-1 (fractions 4–7 harvested from the top). The anti-α 2 δ-1 and anti-α 2 δ-2 antibodies recognize the α 2 -1 and α 2 -2 moieties. ( B ) Aliquots of concentrated DRM fractions from WT (left-hand panel), PrP Tg(WT) (middle panel) and PrP KO (right-hand panel) cerebella were treated with PNGase F and analysed by Western blotting with the indicated antibodies. ( C ) DRM fractions analysed in ( A ) were subjected to PI-PLC treatment and Triton X-114 phase separation (see the Materials and methods section), followed by PNGase F deglycosylation. The proteins remaining in the aqueous (aq) and detergent (det) phase were then resolved on 4–12% Bis-Tris gels and analysed with the indicated antibodies. Lanes 1 and 2 in each panel are from detergent phase fractions, whereas lanes 3 and 4 are from the respective aqueous phase, treated or not with PI-PLC as indicated. ( D ) The PrP Tg(WT) fractions from ( C , middle panel) were also blotted for PrP using the 3F4 antibody. Molecular mass is shown on the right-hand side of the gels in kDa.

    Journal: Biochemical Journal

    Article Title: The inhibition of functional expression of calcium channels by prion protein demonstrates competition with ?2? for GPI-anchoring pathways

    doi: 10.1042/BJ20131405

    Figure Lengend Snippet: PI-PLC treatment and phase separation of α 2 δ-1, α 2 δ-2 and PrP in mouse cerebella ( A ) DRM fractions from WT (left-hand panel), PrP Tg(WT) (middle panel) and PrP KO (right-hand panel) mouse cerebella were prepared as described in the Materials and methods section. Aliquots were resolved on 3–8% Tris/acetate (to resolve α 2 δs) or 4–12% Bis-Tris gels (to resolve PrP in the same samples), and analysed by Western blotting (WB) with relevant antibodies as indicated. The full profile is not shown, but only the fractions of the sucrose gradient corresponding to DRMs identified by the presence of flotillin-1 (fractions 4–7 harvested from the top). The anti-α 2 δ-1 and anti-α 2 δ-2 antibodies recognize the α 2 -1 and α 2 -2 moieties. ( B ) Aliquots of concentrated DRM fractions from WT (left-hand panel), PrP Tg(WT) (middle panel) and PrP KO (right-hand panel) cerebella were treated with PNGase F and analysed by Western blotting with the indicated antibodies. ( C ) DRM fractions analysed in ( A ) were subjected to PI-PLC treatment and Triton X-114 phase separation (see the Materials and methods section), followed by PNGase F deglycosylation. The proteins remaining in the aqueous (aq) and detergent (det) phase were then resolved on 4–12% Bis-Tris gels and analysed with the indicated antibodies. Lanes 1 and 2 in each panel are from detergent phase fractions, whereas lanes 3 and 4 are from the respective aqueous phase, treated or not with PI-PLC as indicated. ( D ) The PrP Tg(WT) fractions from ( C , middle panel) were also blotted for PrP using the 3F4 antibody. Molecular mass is shown on the right-hand side of the gels in kDa.

    Article Snippet: After PI-PLC incubation the samples were supplemented with Triton X-114 (Thermo Scientific) to a final concentration of 1%.

    Techniques: Planar Chromatography, Western Blot

    (A) Dynamic Light Scattering analysis of E2-T1 . E2-T1 protein was dialysed into 50 mM Tris (pH 7.0) containing 0.2% Igepal CA630. The graph shows size distribution by intensity for physical size determination. Six measurements of 30 readings each were performed. (B) Western blot of purified E2-T1. E2-T1 protein was run under non-reducing conditions and probed with mouse sera raised against E2-T1.

    Journal: Microbial Cell Factories

    Article Title: Endotoxin-free purification for the isolation of Bovine Viral Diarrhoea Virus E2 protein from insoluble inclusion body aggregates

    doi: 10.1186/1475-2859-10-57

    Figure Lengend Snippet: (A) Dynamic Light Scattering analysis of E2-T1 . E2-T1 protein was dialysed into 50 mM Tris (pH 7.0) containing 0.2% Igepal CA630. The graph shows size distribution by intensity for physical size determination. Six measurements of 30 readings each were performed. (B) Western blot of purified E2-T1. E2-T1 protein was run under non-reducing conditions and probed with mouse sera raised against E2-T1.

    Article Snippet: The resulting solubilised protein was dialysed at room temperature, with 3 buffer changes over 24 hours against 50 mM Tris (pH 7.0), 0.2% Igepal CA630 (Sigma-Aldrich).

    Techniques: Western Blot, Purification

    A single endogenous HLA-B27 ligand identified from the chlamydial PqqC protein shows identity with the predicted T-cell epitope. A , MALDI-TOF MS spectra of fr. 184 from the HLA-B27-bound peptide pools isolated from the PqqC transfectant ( above ) and untransfected B*2705-C1R cells ( below ) showing a single detected ion peak, at m/z 1055.7, differentially present in the bacterial transfectant. Only the relevant sections of the spectra are shown. This ion peak was also not detected in the adjacent fractions 183 and 185 of the untransfected cells (not shown). The ion peak at m/z 1053.6, labeled with an asterisk , was a shared ligand found in the adjacent fr. 185 from B*2705-C1R. B , MALDI-TOF/TOF MS/MS spectrum of the ion peak at m/z 1055.7 from the bacterial PqqC transfectant showing the assigned sequence ( above ) and MS/MS spectrum of the corresponding synthetic peptide ( below ). The periodically spaced ion peaks in the upper MS/MS spectrum are related to each other by 44 m/z units with a related series differing by 16 m/z units. They arise from traces of the Igepal CA-630 detergent (octylphenyl polyethylene glycol, (C 2 H 4 O)nC 14 H 22 O) used for cell lysis.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Endogenous Processing and Presentation of T-cell Epitopes from Chlamydia trachomatis with Relevance in HLA-B27-associated Reactive Arthritis *

    doi: 10.1074/mcp.M900107-MCP200

    Figure Lengend Snippet: A single endogenous HLA-B27 ligand identified from the chlamydial PqqC protein shows identity with the predicted T-cell epitope. A , MALDI-TOF MS spectra of fr. 184 from the HLA-B27-bound peptide pools isolated from the PqqC transfectant ( above ) and untransfected B*2705-C1R cells ( below ) showing a single detected ion peak, at m/z 1055.7, differentially present in the bacterial transfectant. Only the relevant sections of the spectra are shown. This ion peak was also not detected in the adjacent fractions 183 and 185 of the untransfected cells (not shown). The ion peak at m/z 1053.6, labeled with an asterisk , was a shared ligand found in the adjacent fr. 185 from B*2705-C1R. B , MALDI-TOF/TOF MS/MS spectrum of the ion peak at m/z 1055.7 from the bacterial PqqC transfectant showing the assigned sequence ( above ) and MS/MS spectrum of the corresponding synthetic peptide ( below ). The periodically spaced ion peaks in the upper MS/MS spectrum are related to each other by 44 m/z units with a related series differing by 16 m/z units. They arise from traces of the Igepal CA-630 detergent (octylphenyl polyethylene glycol, (C 2 H 4 O)nC 14 H 22 O) used for cell lysis.

    Article Snippet: Briefly cells were lysed in 1% Igepal CA-630 (Sigma), 20 m m Tris/HCl buffer, 150 m m NaCl, pH 7.5 in the presence of a mixture of protease inhibitors.

    Techniques: Mass Spectrometry, Isolation, Transfection, Labeling, Sequencing, Lysis

    Midi-GAGR binds to FGFR1 and uses FGFR1 signaling pathway to activate CREB and protect neurons from the death caused by oxidative insult. (A) Midi-GAGR- or dextran-conjugated epoxy sepharose beads were mixed with synaptosomal plasma membrane proteins in 0.5% Igepal CA-630 PMEE buffer to pull down midi-GAGR-interacting FGFR1. Precipitated FGFR1 was detected by immunoblotting (n = 2, four rat brains). (B-J) Mouse cortical neurons (DIV4) were pre-treated with H 2 O (vehicle, B,C) or the inhibitors of FGFR1 (SU5402 [SU], 4 μM, D), PKC (staurosporine [Stau], 3 nM, E), MEK (U0126 [U01], 10 μM, F), PI3K (LY294002 [LY], 20 μM, G), CaMKII (KN-62 [KN], 10 μM, H), or PF-573228 (PF, 1 μM, I) for 6 h and then with mock (B) or 1 μM midi-GAGR (+midi, C-I) for 48 h. Neurons were then immunostained with the antibodies to α-tubulin (red) and p-CREB (green). Scale bar = 100 μm. (J) Bar graphs show the average intensities of pCREB after different treatments (n = 60 neurons, mean ± SEM). *, p

    Journal: PLoS ONE

    Article Title: BBB-Permeable, Neuroprotective, and Neurotrophic Polysaccharide, Midi-GAGR

    doi: 10.1371/journal.pone.0149715

    Figure Lengend Snippet: Midi-GAGR binds to FGFR1 and uses FGFR1 signaling pathway to activate CREB and protect neurons from the death caused by oxidative insult. (A) Midi-GAGR- or dextran-conjugated epoxy sepharose beads were mixed with synaptosomal plasma membrane proteins in 0.5% Igepal CA-630 PMEE buffer to pull down midi-GAGR-interacting FGFR1. Precipitated FGFR1 was detected by immunoblotting (n = 2, four rat brains). (B-J) Mouse cortical neurons (DIV4) were pre-treated with H 2 O (vehicle, B,C) or the inhibitors of FGFR1 (SU5402 [SU], 4 μM, D), PKC (staurosporine [Stau], 3 nM, E), MEK (U0126 [U01], 10 μM, F), PI3K (LY294002 [LY], 20 μM, G), CaMKII (KN-62 [KN], 10 μM, H), or PF-573228 (PF, 1 μM, I) for 6 h and then with mock (B) or 1 μM midi-GAGR (+midi, C-I) for 48 h. Neurons were then immunostained with the antibodies to α-tubulin (red) and p-CREB (green). Scale bar = 100 μm. (J) Bar graphs show the average intensities of pCREB after different treatments (n = 60 neurons, mean ± SEM). *, p

    Article Snippet: Primary mouse neurons were dissected from 16 mouse embryos (E17) and plated in the wells of 6-well plates (1 x 106 cells/well), differentiated for 6 days, and treated with polysaccharides for 48 h. Then, neurons were harvested and lysed in 1% Igepal CA-630 (Sigma)-containing PMEE buffer plus protease and phosphatase inhibitor cocktails (Sigma) for protein extraction.

    Techniques:

    MAPK15 participates in the ULK1 complex. A,  HEK293 and NIH3T3 were harvested and lysed in 1% Nonidet P-40 lysis buffer. The proteins (10 mg per sample) were then subjected to immunoprecipitation ( IP ) with isotype control IgG ( IgG ) or MAPK15-specific antibody (custom preparation). Total lysates and immunoprecipitated complexes were subjected to Western blotting ( WB ) to detect endogenous ULK1 and MAPK15 protein levels. Representative images from three different experiments are shown ( n  = 3).  B , HEK293T cells were transfected with plasmid encoding for ULK1-FLAG in combination with HA-MAPK15 WT , HA-MAPK15 KD , or the empty vector. After 24 h, total lysates were subject to anti-FLAG immunoprecipitation and analyzed by Western blot analysis ( n  = 3).  C , HEK293T cells were transfected with different plasmids encoding for FLAG-tagged ULK1, ATG13, FIP200, or ATG101 proteins in combination with HA-MAPK15 or the empty vector. After 24 h, total lysates were subject to anti-FLAG immunoprecipitation and analyzed by Western blot analysis ( n  = 3).  D , HeLa cells stably expressing EGFP-GABARAP were transfected with HA-MAPK15 and ULK1-FLAG plasmids and subjected to immunofluorescence analysis after 48 h. Representative images are from three different experiments ( n  = 3). EGFP-GABARAP is visualized in  green , ULK1-FLAG in  red , and HA-MAPK15 in  blue  ( upper panels ).  White squares  indicate zoom areas.  Scale bars , 10 μm.  E,  colocalization rate was measured by LAS AF (Leica Microsystem) software, analyzing single cells that express both MAPK15 and ULK1 in addition to EGFP-GABARAP. Thresholds were set at 20% for each channel as suggested from the manufacturer's protocol. Reciprocal colocalization rate between ULK1 and GABARAP, ULK1 and MAPK15, and GABARAP and MAPK15 were evaluated. Measures were obtained by analyzing at least 100 cells/sample from three different experiments.  F , colocalization spots were analyzed for fluorescent signal intensity for each channel; the  yellow line  indicates the measured points on the  merged image .

    Journal: The Journal of Biological Chemistry

    Article Title: MAPK15 is part of the ULK complex and controls its activity to regulate early phases of the autophagic process

    doi: 10.1074/jbc.RA118.002527

    Figure Lengend Snippet: MAPK15 participates in the ULK1 complex. A, HEK293 and NIH3T3 were harvested and lysed in 1% Nonidet P-40 lysis buffer. The proteins (10 mg per sample) were then subjected to immunoprecipitation ( IP ) with isotype control IgG ( IgG ) or MAPK15-specific antibody (custom preparation). Total lysates and immunoprecipitated complexes were subjected to Western blotting ( WB ) to detect endogenous ULK1 and MAPK15 protein levels. Representative images from three different experiments are shown ( n = 3). B , HEK293T cells were transfected with plasmid encoding for ULK1-FLAG in combination with HA-MAPK15 WT , HA-MAPK15 KD , or the empty vector. After 24 h, total lysates were subject to anti-FLAG immunoprecipitation and analyzed by Western blot analysis ( n = 3). C , HEK293T cells were transfected with different plasmids encoding for FLAG-tagged ULK1, ATG13, FIP200, or ATG101 proteins in combination with HA-MAPK15 or the empty vector. After 24 h, total lysates were subject to anti-FLAG immunoprecipitation and analyzed by Western blot analysis ( n = 3). D , HeLa cells stably expressing EGFP-GABARAP were transfected with HA-MAPK15 and ULK1-FLAG plasmids and subjected to immunofluorescence analysis after 48 h. Representative images are from three different experiments ( n = 3). EGFP-GABARAP is visualized in green , ULK1-FLAG in red , and HA-MAPK15 in blue ( upper panels ). White squares indicate zoom areas. Scale bars , 10 μm. E, colocalization rate was measured by LAS AF (Leica Microsystem) software, analyzing single cells that express both MAPK15 and ULK1 in addition to EGFP-GABARAP. Thresholds were set at 20% for each channel as suggested from the manufacturer's protocol. Reciprocal colocalization rate between ULK1 and GABARAP, ULK1 and MAPK15, and GABARAP and MAPK15 were evaluated. Measures were obtained by analyzing at least 100 cells/sample from three different experiments. F , colocalization spots were analyzed for fluorescent signal intensity for each channel; the yellow line indicates the measured points on the merged image .

    Article Snippet: Briefly, washed cellular pellet fractions were resuspended in MAPK lysis buffer made of 20 m m HEPES (PAA, ), pH 7.5, 10 m m EGTA (Sigma, E4378), 40 m m β-glycerophosphate (Sigma, G6501), 1% Nonidet P-40 (Sigma, I3021), 2.5 m m MgCl2 (Sigma, M2670), 2 m m orthovanadate (Sigma, S6508), 2 m m NaF (Carlo Erba, 7681494), 1 m m DTT (IBI, IB21040), and protease inhibitors mixture (Roche Diagnostics, 05056489001).

    Techniques: Lysis, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Stable Transfection, Expressing, Immunofluorescence, Software

    Ubiquitination of ATG14L by ZBTB16 and Cullin3. ( A ) 293T cells were transfected with expression vectors of FLAG-ZBTB16 and Myc-Atg14L and cultured for 24 hr. The cells were treated with MG132 (10 µM) for the last 4 hr before harvesting and then lysed in NP-40 buffer. The lysates were immunoprecipitated with anti-ATG14L antibody, and the immunocomplexes were analyzed by western blotting using anti-Flag antibody. The data are expressed as the mean of 3 biological replicates (mean ± SD). Statistical significance was determined by a two-tailed, unpaired Student's t-test. p

    Journal: eLife

    Article Title: G-protein-coupled receptors regulate autophagy by ZBTB16-mediated ubiquitination and proteasomal degradation of Atg14L

    doi: 10.7554/eLife.06734

    Figure Lengend Snippet: Ubiquitination of ATG14L by ZBTB16 and Cullin3. ( A ) 293T cells were transfected with expression vectors of FLAG-ZBTB16 and Myc-Atg14L and cultured for 24 hr. The cells were treated with MG132 (10 µM) for the last 4 hr before harvesting and then lysed in NP-40 buffer. The lysates were immunoprecipitated with anti-ATG14L antibody, and the immunocomplexes were analyzed by western blotting using anti-Flag antibody. The data are expressed as the mean of 3 biological replicates (mean ± SD). Statistical significance was determined by a two-tailed, unpaired Student's t-test. p

    Article Snippet: Immunoprecipitation Cells were lysed with NP-40 buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40, protease inhibitors cocktail [Sigma], 5% glycerol, 10 mM NaF, 1 mM PMSF), or Buffer II (1 mM EDTA, 0.1% NP-40, 10 mM Tris-HCl pH 7.5).

    Techniques: Transfection, Expressing, Cell Culture, Immunoprecipitation, Western Blot, Two Tailed Test

    Phosphorylation of S184/T282 of ZBTB16 by GSK3b is functionally important for regulating Atg14L under serum starvation condition to promote autophagy. ( A ) The expression vectors of Flag-tagged wild type or mutants ZBTB16 and HA-Ub were transfected into HeLa cells and cultured for 24 hr. HeLa cells were treated with or without serum-deprivation (SD) for 4 hr. The cell lysates were collected and subjected to immunoprecipitation with anti-Flag antibody. The immunocomplexes were analyzed by western blotting using anti-HA for ubiquitin or anti-Flag as indicated. ( B ) 293T cells were transfected with the expression vectors of wide-type FLAG-ZBTB16, mutant FLAG-ZBTB16, HA-Ub as indicated and cultured for 20 hr. The cells were then treated with or without MG132 for 4 hr. Fully denatured lysates were diluted with 0.5% NP-40 lysis buffer and IP with anti-Flag antibody. The lysates were WB with indicated antibodies. ( C ) 293T cells were transfected with the expression vectors of wide-type FLAG-ZBTB16, mutant FLAG-ZBTB16, HA-Ub, Myc-ATG14, HA-ROC1, Myc-Cul3 as indicated and cultured for 36 hr. The cells were then treated with MG132 for 2 hr. Fully denatured lysates were diluted with 0.5% NP-40 lysis buffer and IP with anti-ATG14L antibody. The lysates were WB with indicated antibodies. ( D ) HeLa cells were transfected with expression vectors of wide-type FLAG-ZBTB16, mutant FLAG-ZBTB16, Myc-ATG14 as indicated and then lysed in NP-40 buffer. The lysates were immunoprecipitated with anti-Flag antibody, and the immunocomplexes were analyzed by western blotting using anti-Myc antibody. ( E ) The expression vectors of Flag-tagged wild type or ZBTB16 point mutants were transfected into HeLa cells and cultured for 12 hr. The cells were cultured in serum-free condition for indicated periods of time. The cell lysates were then harvested and analyzed by western blotting using indicated antibodies. DOI: http://dx.doi.org/10.7554/eLife.06734.010

    Journal: eLife

    Article Title: G-protein-coupled receptors regulate autophagy by ZBTB16-mediated ubiquitination and proteasomal degradation of Atg14L

    doi: 10.7554/eLife.06734

    Figure Lengend Snippet: Phosphorylation of S184/T282 of ZBTB16 by GSK3b is functionally important for regulating Atg14L under serum starvation condition to promote autophagy. ( A ) The expression vectors of Flag-tagged wild type or mutants ZBTB16 and HA-Ub were transfected into HeLa cells and cultured for 24 hr. HeLa cells were treated with or without serum-deprivation (SD) for 4 hr. The cell lysates were collected and subjected to immunoprecipitation with anti-Flag antibody. The immunocomplexes were analyzed by western blotting using anti-HA for ubiquitin or anti-Flag as indicated. ( B ) 293T cells were transfected with the expression vectors of wide-type FLAG-ZBTB16, mutant FLAG-ZBTB16, HA-Ub as indicated and cultured for 20 hr. The cells were then treated with or without MG132 for 4 hr. Fully denatured lysates were diluted with 0.5% NP-40 lysis buffer and IP with anti-Flag antibody. The lysates were WB with indicated antibodies. ( C ) 293T cells were transfected with the expression vectors of wide-type FLAG-ZBTB16, mutant FLAG-ZBTB16, HA-Ub, Myc-ATG14, HA-ROC1, Myc-Cul3 as indicated and cultured for 36 hr. The cells were then treated with MG132 for 2 hr. Fully denatured lysates were diluted with 0.5% NP-40 lysis buffer and IP with anti-ATG14L antibody. The lysates were WB with indicated antibodies. ( D ) HeLa cells were transfected with expression vectors of wide-type FLAG-ZBTB16, mutant FLAG-ZBTB16, Myc-ATG14 as indicated and then lysed in NP-40 buffer. The lysates were immunoprecipitated with anti-Flag antibody, and the immunocomplexes were analyzed by western blotting using anti-Myc antibody. ( E ) The expression vectors of Flag-tagged wild type or ZBTB16 point mutants were transfected into HeLa cells and cultured for 12 hr. The cells were cultured in serum-free condition for indicated periods of time. The cell lysates were then harvested and analyzed by western blotting using indicated antibodies. DOI: http://dx.doi.org/10.7554/eLife.06734.010

    Article Snippet: Immunoprecipitation Cells were lysed with NP-40 buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% NP-40, protease inhibitors cocktail [Sigma], 5% glycerol, 10 mM NaF, 1 mM PMSF), or Buffer II (1 mM EDTA, 0.1% NP-40, 10 mM Tris-HCl pH 7.5).

    Techniques: Expressing, Transfection, Cell Culture, Immunoprecipitation, Western Blot, Mutagenesis, Lysis

    Antibodies to GW182-immunoprecipitated Ago2, Dicer and XRN1 proteins from HeLa cell extracts as examined by WB analysis. GW182 and associated proteins were immunoprecipitated using human sera 18033 containing antibodies to GW182 coupled with Protein A sepharose beads and by mouse anti-GW182 monoclonal 4B6 coupled to Protein G sepharose beads. Normal human serum (NHS), IC6 human serum and mouse monoclonal antibody to golgin 97 were used as IP controls. Immunopreciptates were carried out with buffer that did not contain detergent. Proteins (40 μg) were resolved on a 10% SDS-PAGE gel and were detected with a 4B6 monoclonal antibody ( a ) or 18033 human anti-GWB serum ( b ). Immunopreciptates were carried out with buffer that contained 0.3% NP-40 detergent. Proteins (40 μg) were resolved on a 6.5% SDS-PAGE gel and were detected using a mouse anti-Ago2 monoclonal 4F9 antibody ( c ). Protein (40 μg) from whole cell extracts or 18033 immunoprecipitates carried out using 0.3% NP-40 buffer were separated on a 6.5% SDS-PAGE gel and probed with NHS or antibodies to golgin 97, GW182, Ago2, XRN1 or Dicer. ( d .

    Journal: Nature protocols

    Article Title: Optimization of immunoprecipitation-western blot analysis in detecting GW182-associated components of GW/P bodies

    doi: 10.1038/nprot.2009.34

    Figure Lengend Snippet: Antibodies to GW182-immunoprecipitated Ago2, Dicer and XRN1 proteins from HeLa cell extracts as examined by WB analysis. GW182 and associated proteins were immunoprecipitated using human sera 18033 containing antibodies to GW182 coupled with Protein A sepharose beads and by mouse anti-GW182 monoclonal 4B6 coupled to Protein G sepharose beads. Normal human serum (NHS), IC6 human serum and mouse monoclonal antibody to golgin 97 were used as IP controls. Immunopreciptates were carried out with buffer that did not contain detergent. Proteins (40 μg) were resolved on a 10% SDS-PAGE gel and were detected with a 4B6 monoclonal antibody ( a ) or 18033 human anti-GWB serum ( b ). Immunopreciptates were carried out with buffer that contained 0.3% NP-40 detergent. Proteins (40 μg) were resolved on a 6.5% SDS-PAGE gel and were detected using a mouse anti-Ago2 monoclonal 4F9 antibody ( c ). Protein (40 μg) from whole cell extracts or 18033 immunoprecipitates carried out using 0.3% NP-40 buffer were separated on a 6.5% SDS-PAGE gel and probed with NHS or antibodies to golgin 97, GW182, Ago2, XRN1 or Dicer. ( d .

    Article Snippet: Colloidal Blue Staining Kit (Invitrogen, cat. no. LC6025) Tris-HCl (Trizma; Sigma, cat. no. T-3253) Tris-base (Bio-Rad, cat. no. 161-0719) Sodium chloride (NaCl; VWR, cat. no. CA-EM7760) 0.5 M EDTA, pH 8.0 (GIBCO/Invitrogen, cat. no. 15575-020) NP-40 detergent (Sigma, cat. no. N-6507) 2× LSB (see REAGENT SETUP) Glycerol (VWR, cat. no. CA-EM4750) Bromophenol blue (VWR, cat. no. CA-EM2830) 2-Mercaptoethanol (Bio-Rad, cat. no. 161-0710) !

    Techniques: Immunoprecipitation, Western Blot, SDS Page

    Immuno-detection of PhoA in fractionated or trypsin treated cellular proteins. A . Triton X-114 partitioning of  M. gallisepticum   cell proteins. Proteins of pTAP or pTP transformed cells were separated into hydrophobic and aqueous fractions by Triton X-114 partitioning, Western transferred and probed with a MAb to alkaline phosphatase. Panel TAP,  M. gallisepticum   transformed with pTAP and expressing PhoA. Panel  A ,  M. gallisepticum   transformed with pTP cells. Lanes W, whole-cells; H, hydrophobic fraction; A, aqueous fraction.  B . Immunostaining of cytosolic and membrane fractions of mycoplasma transformants expressing alkaline phosphatase. The fractions were separated on 10 % SDS-polyacrylamide gels, Western transferred and immunostained using a MAb to alkaline phosphatase. Lanes W, whole cells; M, membrane fraction and C, cytosolic fraction.  C . Surface proteolysis of PhoA. Whole pTAP transformant cells were treated with increasing concentrations of trypsin, the proteins then separated on 10 % SDS-polyacrylamide gels, Western transferred and immunostained using a MAb to AP. Trypsin concentrations (μg/ml) are indicated above each lane. Panels CB, Coomassie brilliant blue stained; WB, Western blot probed with MAb to AP. The arrow indicates the 67 kDa VlhA, which was degraded by increasing concentrations of trypsin. The tryptic products of VlhA can also be seen. Most cellular proteins were minimally affected.

    Journal: BMC Microbiology

    Article Title: A novel transposon construct expressing PhoA with potential for studying protein expression and translocation in Mycoplasma gallisepticum

    doi: 10.1186/1471-2180-12-138

    Figure Lengend Snippet: Immuno-detection of PhoA in fractionated or trypsin treated cellular proteins. A . Triton X-114 partitioning of M. gallisepticum cell proteins. Proteins of pTAP or pTP transformed cells were separated into hydrophobic and aqueous fractions by Triton X-114 partitioning, Western transferred and probed with a MAb to alkaline phosphatase. Panel TAP, M. gallisepticum transformed with pTAP and expressing PhoA. Panel A , M. gallisepticum transformed with pTP cells. Lanes W, whole-cells; H, hydrophobic fraction; A, aqueous fraction. B . Immunostaining of cytosolic and membrane fractions of mycoplasma transformants expressing alkaline phosphatase. The fractions were separated on 10 % SDS-polyacrylamide gels, Western transferred and immunostained using a MAb to alkaline phosphatase. Lanes W, whole cells; M, membrane fraction and C, cytosolic fraction. C . Surface proteolysis of PhoA. Whole pTAP transformant cells were treated with increasing concentrations of trypsin, the proteins then separated on 10 % SDS-polyacrylamide gels, Western transferred and immunostained using a MAb to AP. Trypsin concentrations (μg/ml) are indicated above each lane. Panels CB, Coomassie brilliant blue stained; WB, Western blot probed with MAb to AP. The arrow indicates the 67 kDa VlhA, which was degraded by increasing concentrations of trypsin. The tryptic products of VlhA can also be seen. Most cellular proteins were minimally affected.

    Article Snippet: Partitioning of mycoplasma cell proteins into hydrophobic and aqueous fractions using Triton X-114 Mycoplasma cell proteins from a 20 ml overnight culture were separated into hydrophobic and aqueous fractions using the detergent Triton X-114 (Sigma) [ , ].

    Techniques: Transformation Assay, Western Blot, Expressing, Immunostaining, Staining

    Thy-1 in the Conditioned Media of Normal Human Lung Fibroblasts is insoluble (A)  CM was collected from CCL-210 provided either MEM or 20ng/mL TNFα and IL-1β in MEM for 24 to 48 hrs. Using Triton X-114, concentrated CM was partitioned into an aqueous (AP) and insoluble phase (IP). Mature Thy-1 detected by western blot in the CE of NHLF or partitioned and pre-partitioned CM by western blot using K117.  (B)  CM was collected from CCL-210 provided MEM 48 hrs. CM was submitted to differential centrifugation of 1,200 × g and 21,000 × g then precipitated with methanol. Mature Thy-1 was detected in the precipitated material of total (T) prior to, supernatant (S) after, and pellet (P) after 21,000 × g by western blot using K117.

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Effect of the GPI anchor of human Thy-1 on antibody recognition and function

    doi: 10.1038/labinvest.2012.178

    Figure Lengend Snippet: Thy-1 in the Conditioned Media of Normal Human Lung Fibroblasts is insoluble (A) CM was collected from CCL-210 provided either MEM or 20ng/mL TNFα and IL-1β in MEM for 24 to 48 hrs. Using Triton X-114, concentrated CM was partitioned into an aqueous (AP) and insoluble phase (IP). Mature Thy-1 detected by western blot in the CE of NHLF or partitioned and pre-partitioned CM by western blot using K117. (B) CM was collected from CCL-210 provided MEM 48 hrs. CM was submitted to differential centrifugation of 1,200 × g and 21,000 × g then precipitated with methanol. Mature Thy-1 was detected in the precipitated material of total (T) prior to, supernatant (S) after, and pellet (P) after 21,000 × g by western blot using K117.

    Article Snippet: Partitioning of Conditioned Media using Triton X-114 Triton X-114 (Sigma-Aldrich 93422) was added to concentrated CM to a final concentration of 2%.

    Techniques: Western Blot, Centrifugation

    The dentilisin complex associates only with OM-MOSP. ( A) SDS-PAGE and immunoblot analysis of TX-114 phase partitioned T . denticola cells using antisera directed against PrtP, PrcA1, PrcA2, and PrcB. Lanes: whole cells (WC), TX-114 insoluble material (Ins), aqueous (Aq) and detergent (Det) phase. (B) SDS-PAGE gels of the NP-40 supernatant without (−) or with (+) boiling followed by immunoblot analysis using the same antisera as in Panel A. (C) Eluates from Co-IP of OM and periplasmic conformers were immunoblotted with antisera against the four dentilisin components. (D) SDS-PAGE and immunoblot analysis of eluate when 2% DDM was added to the NP-40 supernatant prior to Co-IP. Molecular mass standards (kDa) are indicated on the left of each gel.

    Journal: Scientific Reports

    Article Title: The major outer sheath protein forms distinct conformers and multimeric complexes in the outer membrane and periplasm of Treponema denticola

    doi: 10.1038/s41598-017-13550-6

    Figure Lengend Snippet: The dentilisin complex associates only with OM-MOSP. ( A) SDS-PAGE and immunoblot analysis of TX-114 phase partitioned T . denticola cells using antisera directed against PrtP, PrcA1, PrcA2, and PrcB. Lanes: whole cells (WC), TX-114 insoluble material (Ins), aqueous (Aq) and detergent (Det) phase. (B) SDS-PAGE gels of the NP-40 supernatant without (−) or with (+) boiling followed by immunoblot analysis using the same antisera as in Panel A. (C) Eluates from Co-IP of OM and periplasmic conformers were immunoblotted with antisera against the four dentilisin components. (D) SDS-PAGE and immunoblot analysis of eluate when 2% DDM was added to the NP-40 supernatant prior to Co-IP. Molecular mass standards (kDa) are indicated on the left of each gel.

    Article Snippet: Triton X-114 phase partitioning Triton X-114 (TX-114) (Sigma-Aldrich, USA) phase partitioning was performed as described previously , , .

    Techniques: SDS Page, Co-Immunoprecipitation Assay

    Dentilisin associates predominantly with the larger OM-MOSP complex. BN-PAGE of the NP-40 supernatant containing the OM-MOSP immunoblotted with rat antiserum directed against MOSP Fl and rabbit antisera against PrtP, PrcA1, PrcA2 and PrcB. Molecular mass standards (kDa) are indicated on the right of each gel.

    Journal: Scientific Reports

    Article Title: The major outer sheath protein forms distinct conformers and multimeric complexes in the outer membrane and periplasm of Treponema denticola

    doi: 10.1038/s41598-017-13550-6

    Figure Lengend Snippet: Dentilisin associates predominantly with the larger OM-MOSP complex. BN-PAGE of the NP-40 supernatant containing the OM-MOSP immunoblotted with rat antiserum directed against MOSP Fl and rabbit antisera against PrtP, PrcA1, PrcA2 and PrcB. Molecular mass standards (kDa) are indicated on the right of each gel.

    Article Snippet: Triton X-114 phase partitioning Triton X-114 (TX-114) (Sigma-Aldrich, USA) phase partitioning was performed as described previously , , .

    Techniques: Polyacrylamide Gel Electrophoresis

    OM- and periplasmic-MOSP form SDS-stable trimers and distinct multimeric complexes. ( A) Protocol outlining the use of NP-40 to separate OM and periplasmic conformers. (B) Immunoblot of the NP-40 soluble (supernatant; NP-40 Sup) and insoluble (pellet; NP40 Pel) fractions obtained by ultracentrifugation which were then separately subjected to TX-114 phase partitioning (TX-114 pp). (C) Immunoblot of BN-PAGE gels of NP-40 supernatant and TX-114 aqueous phase using anti-MOSP Fl antiserum. Also shown are SDS-PAGE gels of the separated conformers without (−) and with (+) boiling followed by immunoblot analysis with anti-MOSP Fl . Molecular mass standards (kDa) are indicated on the left (SDS-PAGE) or right (BN-PAGE) of each gel.

    Journal: Scientific Reports

    Article Title: The major outer sheath protein forms distinct conformers and multimeric complexes in the outer membrane and periplasm of Treponema denticola

    doi: 10.1038/s41598-017-13550-6

    Figure Lengend Snippet: OM- and periplasmic-MOSP form SDS-stable trimers and distinct multimeric complexes. ( A) Protocol outlining the use of NP-40 to separate OM and periplasmic conformers. (B) Immunoblot of the NP-40 soluble (supernatant; NP-40 Sup) and insoluble (pellet; NP40 Pel) fractions obtained by ultracentrifugation which were then separately subjected to TX-114 phase partitioning (TX-114 pp). (C) Immunoblot of BN-PAGE gels of NP-40 supernatant and TX-114 aqueous phase using anti-MOSP Fl antiserum. Also shown are SDS-PAGE gels of the separated conformers without (−) and with (+) boiling followed by immunoblot analysis with anti-MOSP Fl . Molecular mass standards (kDa) are indicated on the left (SDS-PAGE) or right (BN-PAGE) of each gel.

    Article Snippet: Triton X-114 phase partitioning Triton X-114 (TX-114) (Sigma-Aldrich, USA) phase partitioning was performed as described previously , , .

    Techniques: Polyacrylamide Gel Electrophoresis, SDS Page

    Standard curves of phenol–sulfuric acid method conducted at an ice-cold temperature (A) and room temperature (B). (A) -×-: sucrose; ⋯+⋯: sucrose + Triton X-114 (0.05%, w/v); ⋯□⋯: sucrose + Triton X-114 (0.5%, w/v); ⋯○⋯: sucrose + Triton X-114 (5%, w/v); (B) -♦-: sucrose; -■-: sucrose + Triton X-114 (0.5%, w/v); -▴- sucrose + Triton X-100 (0.5%, w/v); -●-: sucrose + EO50PO50 (0.5%, w/v)

    Journal: MethodsX

    Article Title: Colorimetric quantification of sucrose in presence of thermo-sensitive polymers present in aqueous two-phase systems

    doi: 10.1016/j.mex.2014.09.006

    Figure Lengend Snippet: Standard curves of phenol–sulfuric acid method conducted at an ice-cold temperature (A) and room temperature (B). (A) -×-: sucrose; ⋯+⋯: sucrose + Triton X-114 (0.05%, w/v); ⋯□⋯: sucrose + Triton X-114 (0.5%, w/v); ⋯○⋯: sucrose + Triton X-114 (5%, w/v); (B) -♦-: sucrose; -■-: sucrose + Triton X-114 (0.5%, w/v); -▴- sucrose + Triton X-100 (0.5%, w/v); -●-: sucrose + EO50PO50 (0.5%, w/v)

    Article Snippet: Materials • EO50PO50 (Sigma, Cat. No. 438189) • Phenol (Scharlau, Cat. No. FE04821000) • Sucrose (Sigma, Cat. No. S5106) • Sulfuric acid (Merck, Cat. No. 1.00731.1000) • Triton X-100 (Sigma, Cat. No. T9284) • Triton X-114 (Sigma, Cat. No. X144) Ultra-pure water was used throughout the experiment.

    Techniques:

    Sialylated mucins are included in lipid rafts whereas TS is not. (A) Cold Triton X-100 partition. Sialylated trypomastigotes were lysed at 4°C. Mucins were predominantly recovered in the pellet whereas TS and HSP70, a cytosolic protein, were recovered in the supernatant. (B) Triton X-114 extraction for GPI-anchored proteins. Parasites were lysed at 4°C and detergent and aqueous phases separated at 37°C and analyzed by Western blots. Mucins and TS partitioned in the detergent phase due to their GPI-anchoring. Glutamate Dehydrogenase, a cytosolic protein, was recovered in the aqueous phase. (C) Purification of DRMs by step-gradient ultracentrifugation. Trypomastigotes were lysed in Triton X-100 at 4°C or 37°C and centrifuged in an Optiprep gradient. Mucins floated to the 35%-5% interface (lane 6) only when lysis was done at 4°C indicating its DRM nature in contrast to TS. (D) Living parasites were sialylated from a Neu5Az donor, then treated for membrane fluidization with 1% diethyl ether in phosphate-buffered saline (PBS) and fixed with  p -formaldehyde (PFA). Doted labeling for TS and mucins was disrupted only after 90sec treatment even showing colocalization.

    Journal: PLoS Pathogens

    Article Title: Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology

    doi: 10.1371/journal.ppat.1005559

    Figure Lengend Snippet: Sialylated mucins are included in lipid rafts whereas TS is not. (A) Cold Triton X-100 partition. Sialylated trypomastigotes were lysed at 4°C. Mucins were predominantly recovered in the pellet whereas TS and HSP70, a cytosolic protein, were recovered in the supernatant. (B) Triton X-114 extraction for GPI-anchored proteins. Parasites were lysed at 4°C and detergent and aqueous phases separated at 37°C and analyzed by Western blots. Mucins and TS partitioned in the detergent phase due to their GPI-anchoring. Glutamate Dehydrogenase, a cytosolic protein, was recovered in the aqueous phase. (C) Purification of DRMs by step-gradient ultracentrifugation. Trypomastigotes were lysed in Triton X-100 at 4°C or 37°C and centrifuged in an Optiprep gradient. Mucins floated to the 35%-5% interface (lane 6) only when lysis was done at 4°C indicating its DRM nature in contrast to TS. (D) Living parasites were sialylated from a Neu5Az donor, then treated for membrane fluidization with 1% diethyl ether in phosphate-buffered saline (PBS) and fixed with p -formaldehyde (PFA). Doted labeling for TS and mucins was disrupted only after 90sec treatment even showing colocalization.

    Article Snippet: Then parasites were lysed for 1h at 4°C in 2ml Triton X-114 extraction buffer (150mM NaCl, 2% Triton X-114, 50mM Tris pH 7.6, protease inhibitor cocktail (Sigma) and 50μM TLCK).

    Techniques: Western Blot, Purification, Lysis, Labeling

    Triton X-114 solubility of different cell-associated and secreted PPAD species.  P .  gingivalis  isolates were cultured in BHI, and the cell and growth medium fractions were separated by centrifugation. Subsequently, both fractions were extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the different detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting with PPAD-specific antibodies. Names of PPAD sorting type I isolates are underlined. Molecular weights of marker proteins and different PPAD species are indicated. The full-length blot is presented in Figure   S1 .

    Journal: Scientific Reports

    Article Title: Dropping anchor: attachment of peptidylarginine deiminase via A-LPS to secreted outer membrane vesicles of Porphyromonas gingivalis

    doi: 10.1038/s41598-018-27223-5

    Figure Lengend Snippet: Triton X-114 solubility of different cell-associated and secreted PPAD species. P . gingivalis isolates were cultured in BHI, and the cell and growth medium fractions were separated by centrifugation. Subsequently, both fractions were extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the different detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting with PPAD-specific antibodies. Names of PPAD sorting type I isolates are underlined. Molecular weights of marker proteins and different PPAD species are indicated. The full-length blot is presented in Figure  S1 .

    Article Snippet: Aqueous two-phase system protein purification To assess modification of the ~75–85-kDa PPAD species with A-LPS, a protein phase separation assay was applied using the non-ionic detergent Triton X-114 (Sigma-Aldrich, St. Louis, USA) .

    Techniques: Solubility, Cell Culture, Centrifugation, Polyacrylamide Gel Electrophoresis, Western Blot, Marker

    A-LPS modification of PPAD in sorting type I and II isolates of  P .  gingivalis . Cells of  P .  gingivalis  sorting type I and II isolates were cultured in BHI and, subsequently, extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting. In panel A immunodetection was performed with an A-LPS-specific monoclonal antibody. Panel B shows a dual-labeling image upon immunodetection with the A-LPS-specific monoclonal antibody (green signal) and a PPAD-specific rabbit antibody (red signal). Bands representing the A-LPS-modified PPAD species of ~75–85-kDa PPAD are boxed in both panels. Names of sorting type I isolates are underlined. Molecular weights of marker proteins are indicated. The full-length blot is presented in Figure   S1 . Please note that the order of ‘A’- and ‘T’-labeled lanes is inversed compared to Fig.   2  as samples were loaded in a different order.

    Journal: Scientific Reports

    Article Title: Dropping anchor: attachment of peptidylarginine deiminase via A-LPS to secreted outer membrane vesicles of Porphyromonas gingivalis

    doi: 10.1038/s41598-018-27223-5

    Figure Lengend Snippet: A-LPS modification of PPAD in sorting type I and II isolates of P . gingivalis . Cells of P . gingivalis sorting type I and II isolates were cultured in BHI and, subsequently, extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting. In panel A immunodetection was performed with an A-LPS-specific monoclonal antibody. Panel B shows a dual-labeling image upon immunodetection with the A-LPS-specific monoclonal antibody (green signal) and a PPAD-specific rabbit antibody (red signal). Bands representing the A-LPS-modified PPAD species of ~75–85-kDa PPAD are boxed in both panels. Names of sorting type I isolates are underlined. Molecular weights of marker proteins are indicated. The full-length blot is presented in Figure  S1 . Please note that the order of ‘A’- and ‘T’-labeled lanes is inversed compared to Fig.  2 as samples were loaded in a different order.

    Article Snippet: Aqueous two-phase system protein purification To assess modification of the ~75–85-kDa PPAD species with A-LPS, a protein phase separation assay was applied using the non-ionic detergent Triton X-114 (Sigma-Aldrich, St. Louis, USA) .

    Techniques: Modification, Cell Culture, Polyacrylamide Gel Electrophoresis, Western Blot, Immunodetection, Labeling, Marker

    Binding assays of deuterium-substituted ( S )- and ( R )-thalidomides with human CRBN TBD. ( a ) Chemical structures of deuterated ( S )- and ( R )-thalidomides, ( S )-D-Thal and ( R )-D-Thal, respectively. Atom numbering is shown in the ( S )-D-Thal chemical structure. The hydrogen atom at the chiral centre C3 atom of the glutarimide moiety is substituted with a deuterium atom. ( b ) Competitive elution assay using thalidomide-immobilized beads coupled with racemic thalidomide. Beads were mixed with extracts from 293 T cells expressing FLAG-HA-CRBN and washed three times with 0.5% NP-40 lysis buffer and bound proteins were eluted with wash buffer containing 1 mM deuterated ( S )- or ( R )-thalidomide (( S )-D-Thal or ( R )-D-Thal), ( S )- or ( R )-thalidomide (( S )-Thal or ( R )-Thal) or DMSO for the indicated time. The eluate was then analyzed by SDS-PAGE and immunoblotting (IB). ( c ) Inhibitory effects of thalidomide enantiomers on auto-ubiquitylation of FH-CRBN were detected in the presence of MG132. Cells were treated with DMSO or the indicated concentrations of ( S )-D-Thal or ( S )-D-Thal for 4 hours prior to harvesting. Full-length blots in ( b ) and ( c ) are presented in Supplementary Fig. 8 .

    Journal: Scientific Reports

    Article Title: Structural basis of thalidomide enantiomer binding to cereblon

    doi: 10.1038/s41598-018-19202-7

    Figure Lengend Snippet: Binding assays of deuterium-substituted ( S )- and ( R )-thalidomides with human CRBN TBD. ( a ) Chemical structures of deuterated ( S )- and ( R )-thalidomides, ( S )-D-Thal and ( R )-D-Thal, respectively. Atom numbering is shown in the ( S )-D-Thal chemical structure. The hydrogen atom at the chiral centre C3 atom of the glutarimide moiety is substituted with a deuterium atom. ( b ) Competitive elution assay using thalidomide-immobilized beads coupled with racemic thalidomide. Beads were mixed with extracts from 293 T cells expressing FLAG-HA-CRBN and washed three times with 0.5% NP-40 lysis buffer and bound proteins were eluted with wash buffer containing 1 mM deuterated ( S )- or ( R )-thalidomide (( S )-D-Thal or ( R )-D-Thal), ( S )- or ( R )-thalidomide (( S )-Thal or ( R )-Thal) or DMSO for the indicated time. The eluate was then analyzed by SDS-PAGE and immunoblotting (IB). ( c ) Inhibitory effects of thalidomide enantiomers on auto-ubiquitylation of FH-CRBN were detected in the presence of MG132. Cells were treated with DMSO or the indicated concentrations of ( S )-D-Thal or ( S )-D-Thal for 4 hours prior to harvesting. Full-length blots in ( b ) and ( c ) are presented in Supplementary Fig. 8 .

    Article Snippet: Extracts were incubated with M2 FLAG magnetic beads (Sigma) in the presence of (S )-D-thalidomide or (R )-D-thalidomide and incubated for 2 h. Following extensive washing three times with 0.5% NP-40 lysis buffer, bound proteins were eluted with free 3x FLAG-peptides (Sigma) and then subjected to immunoblot analysis.

    Techniques: Binding Assay, Expressing, Lysis, SDS Page

    Effect of low-pH treatment on the hydrophobicity of gB. Soluble gB or BSA was added to 2% Triton X-114 that had been adjusted to the indicated pH. Samples were incubated for 10 min at 37°C and were centrifuged at 300 ×  g  for 3 min. The aqueous supernatant phase and detergent phase were collected and diluted 20-fold in PBS. s-gB samples were subjected to immunoprecipitation with antibody to gB, followed by SDS-PAGE and immunoblotting for gB. BSA samples were trichloroacetic acid (TCA) precipitated and analyzed by SDS-PAGE and Coomassie blue staining.

    Journal: Journal of Virology

    Article Title: Low pH-Induced Conformational Change in Herpes Simplex Virus Glycoprotein B ▿

    doi: 10.1128/JVI.02573-09

    Figure Lengend Snippet: Effect of low-pH treatment on the hydrophobicity of gB. Soluble gB or BSA was added to 2% Triton X-114 that had been adjusted to the indicated pH. Samples were incubated for 10 min at 37°C and were centrifuged at 300 × g for 3 min. The aqueous supernatant phase and detergent phase were collected and diluted 20-fold in PBS. s-gB samples were subjected to immunoprecipitation with antibody to gB, followed by SDS-PAGE and immunoblotting for gB. BSA samples were trichloroacetic acid (TCA) precipitated and analyzed by SDS-PAGE and Coomassie blue staining.

    Article Snippet: Little to no gB was detected in the detergent phase, suggesting little hydrophobic interaction of gB with Triton X-114.

    Techniques: Incubation, Immunoprecipitation, SDS Page, Staining

    TX-114 extraction demonstrates hydrophobic modifications to Rab lipid tail  variants. Triton X-114 fractionation generating a detergent-enriched and  aqueous phase was performed as described under MATERIALS AND METHODS on cells  expressing GFP-Ypt1p, -Ypt1p CTIM , -Ypt1p CIIL ,  -Ypt1p C205S , -Ypt1p Δ CC , -Sec4p,  -Sec4p CTIM , -Sec4p CIIL , -Sec4p C214S , and  -Sec4p Δ CC . The detergent-enriched phase was then  subjected to trichoroacetic acid precipitation followed by SDS-PAGE  electrophoresis and Western blotting to detect the GFP-fusion proteins. As a  control, the fractions were probed for the transmembrane protein Snc1/2p.  Relevant protein markers are indicated.

    Journal: Molecular Biology of the Cell

    Article Title: Dual Prenylation Is Required for Rab Protein Localization and Function

    doi: 10.1091/mbc.E02-11-0707

    Figure Lengend Snippet: TX-114 extraction demonstrates hydrophobic modifications to Rab lipid tail variants. Triton X-114 fractionation generating a detergent-enriched and aqueous phase was performed as described under MATERIALS AND METHODS on cells expressing GFP-Ypt1p, -Ypt1p CTIM , -Ypt1p CIIL , -Ypt1p C205S , -Ypt1p Δ CC , -Sec4p, -Sec4p CTIM , -Sec4p CIIL , -Sec4p C214S , and -Sec4p Δ CC . The detergent-enriched phase was then subjected to trichoroacetic acid precipitation followed by SDS-PAGE electrophoresis and Western blotting to detect the GFP-fusion proteins. As a control, the fractions were probed for the transmembrane protein Snc1/2p. Relevant protein markers are indicated.

    Article Snippet: Then 500 μl of phosphate-buffered saline (PBS) containing 2% Triton X-114 with protease inhibitors (1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, and 10 μg pepstatin A) was added to the post-nuclear supernatants.

    Techniques: Fractionation, Expressing, SDS Page, Electrophoresis, Western Blot