polyacrylamide gel electrophoresis analysis  (Millipore)


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  • 99
    Name:
    SYBR Green I nucleic acid gel stain
    Description:

    Catalog Number:
    s9430
    Price:
    None
    Applications:
    A proprietary unsymmetrical cyanine dye which is an ultrasensitive stain for post-electrophoresis staining of dsDNA in agarose or polyacrylamide gels. SYBR Green I can also be used to detect ssDNA and RNA in denaturing agarose/formaldehyde and polyacrylamide/urea gels without any prewashing steps, although the sensitivity is lower (approximately 100 to 300 pg per band using 254 nm epi-illumination). SYBR Green II is recommended for ss-DNA and RNA.SYBR Green I stain has been shown to be much less mutagenic than ethidium bromide in Ames tests. SYBR Green I is also a very sensitive stain for oligonucleotides, allowing for detection of as little as 1-2 ng of a synthetic 24-mer on a 5% polyacrylamide gel, which is 50-100 times greater sensitivity than obtained with ethidium bromide. Staining agarose gels with SYBR Green I does not interfere with the transfer of nucleic acids to membranes or subsequent hybridization in Southern or Northern blot analysis as long as 0.1%-0.3% SDS is included in prehybridization and hybridization buffers to remove the dye.Sigma Life Science is committed to bringing you Greener Alternative Products, which adhere to one or more of The 12 Principles of Greener Chemistry. This product has inherently safer chemistry, compared to the standard use of ethidium bromide for staining. For more information see publication found in related content.
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    Structured Review

    Millipore polyacrylamide gel electrophoresis analysis
    SYBR Green I nucleic acid gel stain

    https://www.bioz.com/result/polyacrylamide gel electrophoresis analysis/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyacrylamide gel electrophoresis analysis - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Attenuated diphtheria toxin mediates siRNA delivery"

    Article Title: Attenuated diphtheria toxin mediates siRNA delivery

    Journal: Science Advances

    doi: 10.1126/sciadv.aaz4848

    siRNAs can be conjugated to a DT. ( A ) a DT was engineered to contain a free cysteine as a functional handle ( a DT-SH), protected by a SUMO tag and purified using a histidine (His) tag. ( B ) a DT was reacted with a PEG cross-linker containing both maleimide and DBCO functional groups to obtain DBCO-modified a DT ( a DT-DBCO). ( C ) The presence of the DBCO modification on a DT was confirmed by measuring the absorbance at 280 nm ( a DT) and 309 nm (DBCO). Curves shown are a DT before modification (blue) and after DBCO modification (red). A 280 , absorbance at 280 nm. ( D ) Azide-modified siRNA was reacted with the DBCO-functionalized a DT to obtain the a DT-siRNA conjugate. RT, room temperature. ( E ) Modification of the a DT with the siRNA was confirmed via polyacrylamide gel electrophoresis (PAGE) stained with Coomassie blue to localize the DT protein. Lane 1 shows the a DT-DBCO starting material ( M w , ~72,000), and lane 2 shows the a DT-siRNA conjugate ( M w , ~90,000) alone with some unreacted starting material. ( F ) Purification of the excess siRNA was confirmed via PAGE stained with GelRed to localize the siRNA. Lane 3 shows the a DT-siRNA conjugate along with unreacted siRNA; lane 4 shows the a DT-siRNA conjugate after nickel column purification, with only a small amount of excess siRNA left over.
    Figure Legend Snippet: siRNAs can be conjugated to a DT. ( A ) a DT was engineered to contain a free cysteine as a functional handle ( a DT-SH), protected by a SUMO tag and purified using a histidine (His) tag. ( B ) a DT was reacted with a PEG cross-linker containing both maleimide and DBCO functional groups to obtain DBCO-modified a DT ( a DT-DBCO). ( C ) The presence of the DBCO modification on a DT was confirmed by measuring the absorbance at 280 nm ( a DT) and 309 nm (DBCO). Curves shown are a DT before modification (blue) and after DBCO modification (red). A 280 , absorbance at 280 nm. ( D ) Azide-modified siRNA was reacted with the DBCO-functionalized a DT to obtain the a DT-siRNA conjugate. RT, room temperature. ( E ) Modification of the a DT with the siRNA was confirmed via polyacrylamide gel electrophoresis (PAGE) stained with Coomassie blue to localize the DT protein. Lane 1 shows the a DT-DBCO starting material ( M w , ~72,000), and lane 2 shows the a DT-siRNA conjugate ( M w , ~90,000) alone with some unreacted starting material. ( F ) Purification of the excess siRNA was confirmed via PAGE stained with GelRed to localize the siRNA. Lane 3 shows the a DT-siRNA conjugate along with unreacted siRNA; lane 4 shows the a DT-siRNA conjugate after nickel column purification, with only a small amount of excess siRNA left over.

    Techniques Used: Functional Assay, Purification, Modification, Polyacrylamide Gel Electrophoresis, Staining, Nickel Column

    Related Articles

    Conjugation Assay:

    Article Title: Attenuated diphtheria toxin mediates siRNA delivery
    Article Snippet: .. Successful conjugation was confirmed by polyacrylamide gel electrophoresis analysis, and conjugation efficiency was quantified using ImageJ software (45% total yield; 80% recovery of DT, 55% siRNA conjugation efficiency). .. Gene knockdown assays—Treatment with a DT-siRNA conjugate aDT-siRNA was mixed with Opti-MEM for a final concentration of 50 nM and added to 24-well Primaria plates coated with PLO/laminin.

    Agarose Gel Electrophoresis:

    Article Title: A DEK Domain-Containing Protein Modulates Chromatin Structure and Function in Arabidopsis [W] [W] [OPEN]
    Article Snippet: .. After proteinase K (#03115887001; Roche) digestion, DNA was precipitated and analyzed on a 0.8% agarose gel in 0.5× TBE (45 mM Tris-borate and 1 mM EDTA) at 2 V/cm for 16 h. The gel was stained with SYBR Green I (#S9430; Sigma-Aldrich). ..

    SYBR Green Assay:

    Article Title: Genotoxic potential of Cotinus coggygria Scop. (Anacardiaceae) stem extract in vivo
    Article Snippet: .. Immediately prior to analysis, the slides were stained with 90 μL of SYBR GREEN I (Sigma-Aldrich, S 9430). .. Comets were visualized and captured with the 40x objective lens of a Leica DMLB fluorescence microscope, attached to a CCD camera.

    Article Title: Identification of ISMyo2, a novel insertion sequence element of IS21 family and its diagnostic potential for detection of Mycobacterium yongonense
    Article Snippet: .. A 10 μl reaction mixture was prepared for each sample, with the following components: 1 μl of Taq buffer (including 20 mM MgCl2 ) supplied together with Ex Taq HS (Takara), 0.25 μM forward primer, 0.25 μM reverse primer, 0.2 mM dNTPs, 0.7 mg/ml BSA (NEB), 0.5 × SYBR Green I (Sigma S9430), 3 % DMSO, 0.25 U of ExTaq HS, and sterile distilled water. .. The cycling conditions were 2 min at 95 °C and 5 s at 98 °C, followed by 35 or 45 cycles of 10 s at 98 °C, 10 s at 64 °C and 40 s at 72 °C (single acquisition of fluorescence signals).

    Article Title: miR-148a-3p regulates alcoholic liver fibrosis through targeting ERBB3
    Article Snippet: .. A volume of 0.5 µ l forward primer (10 µ M), 0.5 µ l reverse primer (10 µ M), 4 µ l cDNA, 5 µ l SYBR Green I nucleic acid gel stain (cat. no. S9430; Sigma-Aldrich; Merck KGaA) were mixed into 10 µ l solution. .. The thermocycler conditions for the qPCR assay was set as follows: Pretreatment at 95°C for 1 min, followed by 40 cycles of 95°C for 30 sec, 58°C for 20 sec and 70°C for 20 sec.

    Article Title: A DEK Domain-Containing Protein Modulates Chromatin Structure and Function in Arabidopsis [W] [W] [OPEN]
    Article Snippet: .. After proteinase K (#03115887001; Roche) digestion, DNA was precipitated and analyzed on a 0.8% agarose gel in 0.5× TBE (45 mM Tris-borate and 1 mM EDTA) at 2 V/cm for 16 h. The gel was stained with SYBR Green I (#S9430; Sigma-Aldrich). ..

    Article Title: Monitoring differentiation of human embryonic stem cells using real-time PCR.
    Article Snippet: .. There is a general lack of rapid, sensitive, and quantitative methods for the detection of differentiating human embryonic stem cells (hESCs). .. There is a general lack of rapid, sensitive, and quantitative methods for the detection of differentiating human embryonic stem cells (hESCs).

    Article Title: Loop mediated isothermal amplification of 5.8S rDNA for specific detection of Tritrichomonas foetus
    Article Snippet: .. For visual inspection, 2 μL of 1/100 dilution of SYBR Green I (S9430, Sigma–Aldrich) were added to 25 μL LAMP reactions. ..

    Article Title: One-Pot Assembly of Complex Giant Unilamellar Vesicle-Based Synthetic Cells
    Article Snippet: .. For image acquisition, released RNA containing GUVs were incubated with 0.5× SYBR Green I (Sigma-Aldrich S9430, Germany) for 30 min and imaged as previously described. .. Polybead Polystyrene Microspheres with a diameter of 1 μm were purchased from Polysciences, Inc. (North America) and added at a concentration of 108 beads/to the aqueous phase for GUV formation.

    Incubation:

    Article Title: One-Pot Assembly of Complex Giant Unilamellar Vesicle-Based Synthetic Cells
    Article Snippet: .. For image acquisition, released RNA containing GUVs were incubated with 0.5× SYBR Green I (Sigma-Aldrich S9430, Germany) for 30 min and imaged as previously described. .. Polybead Polystyrene Microspheres with a diameter of 1 μm were purchased from Polysciences, Inc. (North America) and added at a concentration of 108 beads/to the aqueous phase for GUV formation.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Attenuated diphtheria toxin mediates siRNA delivery
    Article Snippet: .. Successful conjugation was confirmed by polyacrylamide gel electrophoresis analysis, and conjugation efficiency was quantified using ImageJ software (45% total yield; 80% recovery of DT, 55% siRNA conjugation efficiency). .. Gene knockdown assays—Treatment with a DT-siRNA conjugate aDT-siRNA was mixed with Opti-MEM for a final concentration of 50 nM and added to 24-well Primaria plates coated with PLO/laminin.

    Staining:

    Article Title: Genotoxic potential of Cotinus coggygria Scop. (Anacardiaceae) stem extract in vivo
    Article Snippet: .. Immediately prior to analysis, the slides were stained with 90 μL of SYBR GREEN I (Sigma-Aldrich, S 9430). .. Comets were visualized and captured with the 40x objective lens of a Leica DMLB fluorescence microscope, attached to a CCD camera.

    Article Title: miR-148a-3p regulates alcoholic liver fibrosis through targeting ERBB3
    Article Snippet: .. A volume of 0.5 µ l forward primer (10 µ M), 0.5 µ l reverse primer (10 µ M), 4 µ l cDNA, 5 µ l SYBR Green I nucleic acid gel stain (cat. no. S9430; Sigma-Aldrich; Merck KGaA) were mixed into 10 µ l solution. .. The thermocycler conditions for the qPCR assay was set as follows: Pretreatment at 95°C for 1 min, followed by 40 cycles of 95°C for 30 sec, 58°C for 20 sec and 70°C for 20 sec.

    Article Title: A DEK Domain-Containing Protein Modulates Chromatin Structure and Function in Arabidopsis [W] [W] [OPEN]
    Article Snippet: .. After proteinase K (#03115887001; Roche) digestion, DNA was precipitated and analyzed on a 0.8% agarose gel in 0.5× TBE (45 mM Tris-borate and 1 mM EDTA) at 2 V/cm for 16 h. The gel was stained with SYBR Green I (#S9430; Sigma-Aldrich). ..

    Software:

    Article Title: Attenuated diphtheria toxin mediates siRNA delivery
    Article Snippet: .. Successful conjugation was confirmed by polyacrylamide gel electrophoresis analysis, and conjugation efficiency was quantified using ImageJ software (45% total yield; 80% recovery of DT, 55% siRNA conjugation efficiency). .. Gene knockdown assays—Treatment with a DT-siRNA conjugate aDT-siRNA was mixed with Opti-MEM for a final concentration of 50 nM and added to 24-well Primaria plates coated with PLO/laminin.

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  • 93
    Millipore immunoblotting analysis
    Specificity of antihuman l ‐type amino acid transporter 1 ( LAT 1) mA bs in human and macaca cells. A, ACHN human and MK .P3 macaca cells were stained with antihuman LAT 1 mA bs (Ab1 or Ab2), followed by phycoerythrin ( PE )‐conjugated antirat IgG, and analyzed by flow cytometry (FCM). Values indicate the ratio (+ mA b/− mA b) of mean fluorescence intensity ( rMFI ). B, Effects of RNA interference on the expression of human LAT 1 and CD 98 heavy chain ( CD 98hc) proteins in human and macaca cells were analyzed. Cells were treated with mock (scramble) or LAT 1 si RNA (#1, #2 or #3) for 72 h, stained with rat mA bs against human LAT 1 (Ab1) or CD 98hc ( HR 35), followed by PE ‐conjugated antirat IgG, and analyzed for protein expression by FCM . C, Antihuman LAT 1 mA b (Ab1) was evaluated for its reactivity against RH 7777 rat cells expressing macaca (left) or human (right) LAT 1‐ GFP . D, Association of introduced macaca LAT 1‐ GFP with endogenous rat or human CD 98hc was examined. Cell lysates from RH 7777 (upper) or HEK 293 (lower) expressing macaca LAT 1 were subjected to immunoprecipitation with control IgG, anti‐ LAT 1, or anti‐ CD 98hc mA b, and the resulting precipitates, as well as the original cell lysates (input), were subjected to <t>immunoblotting</t> with anti‐GFP rabbit polyclonal antibodies
    Immunoblotting Analysis, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore wb immunoprecipitation analysis equal amounts
    TRAP1 interacts with translation factors and is associated to ribosomes. ( a ), ( b ) and ( c ) Total HCT116 lysates were immunoprecipitated with anti-TRAP1 and anti-eEF1A antibodies and immunoblotted with indicated antibodies. No Ab, total cellular extracts incubated with A/G plus agarose beads without antibody; IP, <t>immunoprecipitation</t> with the corresponding antibodies. ( d ) Separation of cytoplasmic extracts from HCT116 cells was performed by ultracentrifugation on sucrose gradients as described in Materials and Methods. Absorbance profile as well as the signals of ribosomal protein-specific antibodies indicate the fractions containing ribosomal particles (40S, 60S, 80S) whereas larger polysomes were collected in the first fraction. The upper part of the gradient (fractions 9,10,11) includes free cytosolic proteins or light complexes
    Wb Immunoprecipitation Analysis Equal Amounts, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore immunoblotting analysis bacterial pellets
    H tr A protein expression, oligomer formation and secretion in a large set of strains. A. Casein zymography of the indicated H . pylori strains reveals proteolytically active H tr A protein species of 55 kDa , 165 kDa and 220 kDa as labelled with arrows. Purified recombinant H tr A of 26695 is loaded in lane 9 and produced the same pattern of active HtrA forms. B. H . pylori isolates were grown in liquid BHI medium for 12 h and then fractionated in bacterial cell pellets (top) and supernatants (bottom). <t>Immunoblotting</t> using α‐ H tr A antibodies show that all H . pylori strains exhibit strong H tr A signals in the supernatant fraction but varied substantially in their band intensities. As control, the bacterial pellets and supernatants were probed with α‐ CagA and α‐ UreB antibodies, excluding artificially lysed bacteria.
    Immunoblotting Analysis Bacterial Pellets, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/immunoblotting analysis bacterial pellets/product/Millipore
    Average 88 stars, based on 1 article reviews
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    Image Search Results


    Specificity of antihuman l ‐type amino acid transporter 1 ( LAT 1) mA bs in human and macaca cells. A, ACHN human and MK .P3 macaca cells were stained with antihuman LAT 1 mA bs (Ab1 or Ab2), followed by phycoerythrin ( PE )‐conjugated antirat IgG, and analyzed by flow cytometry (FCM). Values indicate the ratio (+ mA b/− mA b) of mean fluorescence intensity ( rMFI ). B, Effects of RNA interference on the expression of human LAT 1 and CD 98 heavy chain ( CD 98hc) proteins in human and macaca cells were analyzed. Cells were treated with mock (scramble) or LAT 1 si RNA (#1, #2 or #3) for 72 h, stained with rat mA bs against human LAT 1 (Ab1) or CD 98hc ( HR 35), followed by PE ‐conjugated antirat IgG, and analyzed for protein expression by FCM . C, Antihuman LAT 1 mA b (Ab1) was evaluated for its reactivity against RH 7777 rat cells expressing macaca (left) or human (right) LAT 1‐ GFP . D, Association of introduced macaca LAT 1‐ GFP with endogenous rat or human CD 98hc was examined. Cell lysates from RH 7777 (upper) or HEK 293 (lower) expressing macaca LAT 1 were subjected to immunoprecipitation with control IgG, anti‐ LAT 1, or anti‐ CD 98hc mA b, and the resulting precipitates, as well as the original cell lysates (input), were subjected to immunoblotting with anti‐GFP rabbit polyclonal antibodies

    Journal: Cancer Science

    Article Title: Anti‐tumor effects of mAb against l‐type amino acid transporter 1 ( LAT1) bound to human and monkey LAT1 with dual avidity modes, et al. Anti‐tumor effects of mAb to l ‐type amino acid transporter 1 (LAT1) bound to human and monkey LAT1 with dual avidity modes

    doi: 10.1111/cas.13908

    Figure Lengend Snippet: Specificity of antihuman l ‐type amino acid transporter 1 ( LAT 1) mA bs in human and macaca cells. A, ACHN human and MK .P3 macaca cells were stained with antihuman LAT 1 mA bs (Ab1 or Ab2), followed by phycoerythrin ( PE )‐conjugated antirat IgG, and analyzed by flow cytometry (FCM). Values indicate the ratio (+ mA b/− mA b) of mean fluorescence intensity ( rMFI ). B, Effects of RNA interference on the expression of human LAT 1 and CD 98 heavy chain ( CD 98hc) proteins in human and macaca cells were analyzed. Cells were treated with mock (scramble) or LAT 1 si RNA (#1, #2 or #3) for 72 h, stained with rat mA bs against human LAT 1 (Ab1) or CD 98hc ( HR 35), followed by PE ‐conjugated antirat IgG, and analyzed for protein expression by FCM . C, Antihuman LAT 1 mA b (Ab1) was evaluated for its reactivity against RH 7777 rat cells expressing macaca (left) or human (right) LAT 1‐ GFP . D, Association of introduced macaca LAT 1‐ GFP with endogenous rat or human CD 98hc was examined. Cell lysates from RH 7777 (upper) or HEK 293 (lower) expressing macaca LAT 1 were subjected to immunoprecipitation with control IgG, anti‐ LAT 1, or anti‐ CD 98hc mA b, and the resulting precipitates, as well as the original cell lysates (input), were subjected to immunoblotting with anti‐GFP rabbit polyclonal antibodies

    Article Snippet: For immunoblotting analysis, proteins were solubilized in loading buffer, subjected to SDS‐PAGE (8% gels) and transferred to Immobilon‐P (Millipore), and exposed to rabbit anti‐GFP pAb.

    Techniques: Staining, Flow Cytometry, Cytometry, Fluorescence, Expressing, Immunoprecipitation

    Decreased p38mapk activation in aortas of obese Arg-II -/- mouse. Immunoblotting analysis of the Thr180/Tyr182 phosphorylated p38mapk (p-p38mapk) and total p38mapk in aortas of WT and Arg-II -/- mice fed HFD. n = 11, ***p

    Journal: Cardiovascular Diabetology

    Article Title: p38 mitogen-activated protein kinase is involved in arginase-II-mediated eNOS-Uncoupling in Obesity

    doi: 10.1186/s12933-014-0113-z

    Figure Lengend Snippet: Decreased p38mapk activation in aortas of obese Arg-II -/- mouse. Immunoblotting analysis of the Thr180/Tyr182 phosphorylated p38mapk (p-p38mapk) and total p38mapk in aortas of WT and Arg-II -/- mice fed HFD. n = 11, ***p

    Article Snippet: Immunoblotting analysis Preparation of mouse aortic tissue and endothelium cell extract, SDS-PAGE, transfer of SDS gels to an Immobilon-P membrane (Millipore) were performed as previously described [[ ]].

    Techniques: Activation Assay, Mouse Assay

    HFD feeding enhances eNOS, Arg-II expression/activity, and p38mapk activation in mouse aortas. (A) Immunoblotting analysis of eNOS total protein and eNOS-S1177 (p-eNOS) levels (n = 5). (B) Immunoblotting analysis of Arg-II (Arg-I not detectable, tubulin served as loading control), p38mapk activation, i.e., Thr180/Tyr182-phosphorylated p38mapk (p-p38mapk) and total p38mapk in the aortas of WT mice fed NC or HFD (n = 5), and arginase activity assay (n = 7-9). *p

    Journal: Cardiovascular Diabetology

    Article Title: p38 mitogen-activated protein kinase is involved in arginase-II-mediated eNOS-Uncoupling in Obesity

    doi: 10.1186/s12933-014-0113-z

    Figure Lengend Snippet: HFD feeding enhances eNOS, Arg-II expression/activity, and p38mapk activation in mouse aortas. (A) Immunoblotting analysis of eNOS total protein and eNOS-S1177 (p-eNOS) levels (n = 5). (B) Immunoblotting analysis of Arg-II (Arg-I not detectable, tubulin served as loading control), p38mapk activation, i.e., Thr180/Tyr182-phosphorylated p38mapk (p-p38mapk) and total p38mapk in the aortas of WT mice fed NC or HFD (n = 5), and arginase activity assay (n = 7-9). *p

    Article Snippet: Immunoblotting analysis Preparation of mouse aortic tissue and endothelium cell extract, SDS-PAGE, transfer of SDS gels to an Immobilon-P membrane (Millipore) were performed as previously described [[ ]].

    Techniques: Expressing, Activity Assay, Activation Assay, Mouse Assay, Arginase Activity Assay

    TRAP1 interacts with translation factors and is associated to ribosomes. ( a ), ( b ) and ( c ) Total HCT116 lysates were immunoprecipitated with anti-TRAP1 and anti-eEF1A antibodies and immunoblotted with indicated antibodies. No Ab, total cellular extracts incubated with A/G plus agarose beads without antibody; IP, immunoprecipitation with the corresponding antibodies. ( d ) Separation of cytoplasmic extracts from HCT116 cells was performed by ultracentrifugation on sucrose gradients as described in Materials and Methods. Absorbance profile as well as the signals of ribosomal protein-specific antibodies indicate the fractions containing ribosomal particles (40S, 60S, 80S) whereas larger polysomes were collected in the first fraction. The upper part of the gradient (fractions 9,10,11) includes free cytosolic proteins or light complexes

    Journal: Cell Death & Disease

    Article Title: Translational control in the stress adaptive response of cancer cells: a novel role for the heat shock protein TRAP1

    doi: 10.1038/cddis.2013.379

    Figure Lengend Snippet: TRAP1 interacts with translation factors and is associated to ribosomes. ( a ), ( b ) and ( c ) Total HCT116 lysates were immunoprecipitated with anti-TRAP1 and anti-eEF1A antibodies and immunoblotted with indicated antibodies. No Ab, total cellular extracts incubated with A/G plus agarose beads without antibody; IP, immunoprecipitation with the corresponding antibodies. ( d ) Separation of cytoplasmic extracts from HCT116 cells was performed by ultracentrifugation on sucrose gradients as described in Materials and Methods. Absorbance profile as well as the signals of ribosomal protein-specific antibodies indicate the fractions containing ribosomal particles (40S, 60S, 80S) whereas larger polysomes were collected in the first fraction. The upper part of the gradient (fractions 9,10,11) includes free cytosolic proteins or light complexes

    Article Snippet: WB/Immunoprecipitation analysis Equal amounts of protein from cell lysates and tumor specimens were subjected to SDS-PAGE and transferred to a PVDF membrane (Millipore).

    Techniques: Immunoprecipitation, Incubation

    TRAP1 regulates eIF2 α -ATF4 pathway. ( a ) Scrambled and sh-TRAP1 HCT116 cells were treated with TG (1 μ M) for the indicated times. Total lysates were immunoblotted with indicated antibodies. ( b ) HCT116 cells were starved for 6 h in cysteine/methionine/glutammine-free medium or low glucose (1.5 mg/ml) medium respectively. Total lysates were immunoblotted with the indicated antibodies. ( c ) TRAP1 and GCN2 co-immunoprecipitate: total HCT116 lysates were immunoprecipitated with anti-TRAP1 and anti-GCN2 antibodies and immunoblotted with indicated antibodies. No Ab, total cellular extracts incubated with A/G plus agarose beads without antibody; IP, immunoprecipitation with the corresponding antibodies. ( d ) TRAP1/GCN2 colocalization: HCT116 cells were fixed and treated as described in Materials and Methods. Panel 1 shows staining of GCN2 (green); panel 2 shows staining of TRAP1 (red); in Panel 3, a double immunofluorescent staining is shown. ( e ) Real-time RT-PCR analysis of BiP/Grp78 mRNA expression in scrambled and sh-TRAP1 HCT116 cells exposed to 1 μ M TG for 12 h or starved for 48 h in low serum or low glucose medium. All data are expressed as mean±S.D. from two independent experiments; * P ≤0.01, ** P ≤0.001, *** P ≤0.0001, n =2. ( f ) Luciferase assay on xCT promoter constructs transfected in sh-TRAP1 HCT116 cells and scrambled control. All data are expressed as mean±S.D. from two independent experiments; * P ≤0.01, ** P ≤0.001, *** P ≤0.0001, n =2

    Journal: Cell Death & Disease

    Article Title: Translational control in the stress adaptive response of cancer cells: a novel role for the heat shock protein TRAP1

    doi: 10.1038/cddis.2013.379

    Figure Lengend Snippet: TRAP1 regulates eIF2 α -ATF4 pathway. ( a ) Scrambled and sh-TRAP1 HCT116 cells were treated with TG (1 μ M) for the indicated times. Total lysates were immunoblotted with indicated antibodies. ( b ) HCT116 cells were starved for 6 h in cysteine/methionine/glutammine-free medium or low glucose (1.5 mg/ml) medium respectively. Total lysates were immunoblotted with the indicated antibodies. ( c ) TRAP1 and GCN2 co-immunoprecipitate: total HCT116 lysates were immunoprecipitated with anti-TRAP1 and anti-GCN2 antibodies and immunoblotted with indicated antibodies. No Ab, total cellular extracts incubated with A/G plus agarose beads without antibody; IP, immunoprecipitation with the corresponding antibodies. ( d ) TRAP1/GCN2 colocalization: HCT116 cells were fixed and treated as described in Materials and Methods. Panel 1 shows staining of GCN2 (green); panel 2 shows staining of TRAP1 (red); in Panel 3, a double immunofluorescent staining is shown. ( e ) Real-time RT-PCR analysis of BiP/Grp78 mRNA expression in scrambled and sh-TRAP1 HCT116 cells exposed to 1 μ M TG for 12 h or starved for 48 h in low serum or low glucose medium. All data are expressed as mean±S.D. from two independent experiments; * P ≤0.01, ** P ≤0.001, *** P ≤0.0001, n =2. ( f ) Luciferase assay on xCT promoter constructs transfected in sh-TRAP1 HCT116 cells and scrambled control. All data are expressed as mean±S.D. from two independent experiments; * P ≤0.01, ** P ≤0.001, *** P ≤0.0001, n =2

    Article Snippet: WB/Immunoprecipitation analysis Equal amounts of protein from cell lysates and tumor specimens were subjected to SDS-PAGE and transferred to a PVDF membrane (Millipore).

    Techniques: Immunoprecipitation, Incubation, Staining, Quantitative RT-PCR, Expressing, Luciferase, Construct, Transfection

    H tr A protein expression, oligomer formation and secretion in a large set of strains. A. Casein zymography of the indicated H . pylori strains reveals proteolytically active H tr A protein species of 55 kDa , 165 kDa and 220 kDa as labelled with arrows. Purified recombinant H tr A of 26695 is loaded in lane 9 and produced the same pattern of active HtrA forms. B. H . pylori isolates were grown in liquid BHI medium for 12 h and then fractionated in bacterial cell pellets (top) and supernatants (bottom). Immunoblotting using α‐ H tr A antibodies show that all H . pylori strains exhibit strong H tr A signals in the supernatant fraction but varied substantially in their band intensities. As control, the bacterial pellets and supernatants were probed with α‐ CagA and α‐ UreB antibodies, excluding artificially lysed bacteria.

    Journal: Molecular Microbiology

    Article Title: Characterisation of worldwide Helicobacter pylori strains reveals genetic conservation and essentiality of serine protease HtrA

    doi: 10.1111/mmi.13276

    Figure Lengend Snippet: H tr A protein expression, oligomer formation and secretion in a large set of strains. A. Casein zymography of the indicated H . pylori strains reveals proteolytically active H tr A protein species of 55 kDa , 165 kDa and 220 kDa as labelled with arrows. Purified recombinant H tr A of 26695 is loaded in lane 9 and produced the same pattern of active HtrA forms. B. H . pylori isolates were grown in liquid BHI medium for 12 h and then fractionated in bacterial cell pellets (top) and supernatants (bottom). Immunoblotting using α‐ H tr A antibodies show that all H . pylori strains exhibit strong H tr A signals in the supernatant fraction but varied substantially in their band intensities. As control, the bacterial pellets and supernatants were probed with α‐ CagA and α‐ UreB antibodies, excluding artificially lysed bacteria.

    Article Snippet: SDS‐PAGE and immunoblotting analysis Bacterial pellets or cleavage reactions were mixed with equal amounts of 2× SDS‐PAGE buffer and boiled for 5 min. Proteins were separated by SDS‐PAGE on 8% polyacrylamide gels and blotted onto PVDF membranes (Immobilon‐P, Millipore, USA) as described (Lind et al ., ).

    Techniques: Expressing, Zymography, Purification, Recombinant, Produced