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Millipore mgcl 2
Mgcl 2, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 21 article reviews
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Staining:

Article Title: Effect of laser-assisted hatching and necrotic blastomere removal on the development of vitrified-warmed four-cell mouse embryos
Article Snippet: .. Fixed and stained blastocysts were then transferred from solution 2 directly into glycerol (Sigma), taking care to avoid carryover of excessive amounts of solution 2. .. Blastocysts were then mounted onto a glass microscope slide in a drop of glycerol, gently flattened with a cover slip, and visualized for cell counting.

Incubation:

Article Title: Effect of laser-assisted hatching and necrotic blastomere removal on the development of vitrified-warmed four-cell mouse embryos
Article Snippet: .. The expanded blastocysts were first incubated in 500 μl of solution 1 (BSA-free human tubal fluid (HTF) medium with 1% Triton X-100 (Sigma) and 100 μg/ml propidium iodide (Sigma) for up to 30 s. Blastocysts were then immediately transferred into 500 μl of solution 2 (fixative solution of 100% ethanol with 25 μg/ml Bisbenzamide (Hoechst 33258, Calbiochem, San Diego, USA) and stored at 4°C overnight. .. Fixed and stained blastocysts were then transferred from solution 2 directly into glycerol (Sigma), taking care to avoid carryover of excessive amounts of solution 2.

Flow Cytometry:

Article Title: Hepatocyte gene expression and DNA methylation as ancestry-dependent mechanisms in African Americans
Article Snippet: .. The liver was washed by perfusion of 1 L Solution 1 (HEPES buffer, Sigma-Aldrich), flow rate 100–400 mL/min, with no recirculation, followed by perfusion with 1 L of Solution 2 (EGTA buffer, Sigma-Aldrich), flow rate 100–400 mL/min, with no recirculation. .. The tissue was washed to remove the EGTA compound by perfusion of 1 L Solution 1, flow rate 100–400 mL/min, with no recirculation.

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  • 99
    Millipore histone demethylase assay buffer
    Histone Demethylase Assay Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Millipore ubiquitination buffer
    The domain encoded by the carboxyl terminus of exon 3 of ICP0 acts as an E3 ligase, whereas the sequences encoded by exon 2 bind cdc34 ubiquitin-conjugating enzyme. ( A ) Immunoblots of electrophoretically separated products of substrate-independent in vitro <t>ubiquitination</t> reactions. GST (lanes 1 and 2), GST-exon 2 (lanes 3 and 4), GST-exon 3 (lanes 5 and 6), and no additional protein (lane 7) were added to the substrate-independent in vitro ubiquitination reaction master mix (MM) containing recombinant Uba1 (E1), recombinant cdc34, biotinylated ubiquitin, and ubiquitination buffer in the presence and absence of ATP and an ATP regenerating system, as described in Materials and Methods . The reaction was stopped after 90 min, and the reaction mixture was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. ( B ) Electrophoretically separated reaction mixtures containing the indicated GST fusion protein in addition to the master mix (lanes 8–13) or the master mix alone (lane 14) in the presence and absence of ATP and an ATP regenerating system were probed with a rabbit polyclonal antibody directed against GST. ( C ) cdc34 was precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 15–17) or the master mix alone (lane 18) in the presence of ATP and an ATP regenerating system. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. ( D ) GST or GST fusion proteins were precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 19–21) or the master mix alone (lane 22) in the presence of ATP and an ATP regenerating system by using glutathione Sepharose beads. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with a rabbit polyclonal antibody directed against cdc34. The dots to the right of high molecular weight bands in lanes 6 and 17 identify ubiquitinated proteins; G, GST; 2, GST-exon 2 chimeric protein; 3, GST-carboxyl-terminal domain of exon 3 fusion protein.
    Ubiquitination Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore kinase buffer
    The domain encoded by the carboxyl terminus of exon 3 of ICP0 acts as an E3 ligase, whereas the sequences encoded by exon 2 bind cdc34 ubiquitin-conjugating enzyme. ( A ) Immunoblots of electrophoretically separated products of substrate-independent in vitro <t>ubiquitination</t> reactions. GST (lanes 1 and 2), GST-exon 2 (lanes 3 and 4), GST-exon 3 (lanes 5 and 6), and no additional protein (lane 7) were added to the substrate-independent in vitro ubiquitination reaction master mix (MM) containing recombinant Uba1 (E1), recombinant cdc34, biotinylated ubiquitin, and ubiquitination buffer in the presence and absence of ATP and an ATP regenerating system, as described in Materials and Methods . The reaction was stopped after 90 min, and the reaction mixture was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. ( B ) Electrophoretically separated reaction mixtures containing the indicated GST fusion protein in addition to the master mix (lanes 8–13) or the master mix alone (lane 14) in the presence and absence of ATP and an ATP regenerating system were probed with a rabbit polyclonal antibody directed against GST. ( C ) cdc34 was precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 15–17) or the master mix alone (lane 18) in the presence of ATP and an ATP regenerating system. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. ( D ) GST or GST fusion proteins were precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 19–21) or the master mix alone (lane 22) in the presence of ATP and an ATP regenerating system by using glutathione Sepharose beads. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with a rabbit polyclonal antibody directed against cdc34. The dots to the right of high molecular weight bands in lanes 6 and 17 identify ubiquitinated proteins; G, GST; 2, GST-exon 2 chimeric protein; 3, GST-carboxyl-terminal domain of exon 3 fusion protein.
    Kinase Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 152 article reviews
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    The domain encoded by the carboxyl terminus of exon 3 of ICP0 acts as an E3 ligase, whereas the sequences encoded by exon 2 bind cdc34 ubiquitin-conjugating enzyme. ( A ) Immunoblots of electrophoretically separated products of substrate-independent in vitro ubiquitination reactions. GST (lanes 1 and 2), GST-exon 2 (lanes 3 and 4), GST-exon 3 (lanes 5 and 6), and no additional protein (lane 7) were added to the substrate-independent in vitro ubiquitination reaction master mix (MM) containing recombinant Uba1 (E1), recombinant cdc34, biotinylated ubiquitin, and ubiquitination buffer in the presence and absence of ATP and an ATP regenerating system, as described in Materials and Methods . The reaction was stopped after 90 min, and the reaction mixture was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. ( B ) Electrophoretically separated reaction mixtures containing the indicated GST fusion protein in addition to the master mix (lanes 8–13) or the master mix alone (lane 14) in the presence and absence of ATP and an ATP regenerating system were probed with a rabbit polyclonal antibody directed against GST. ( C ) cdc34 was precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 15–17) or the master mix alone (lane 18) in the presence of ATP and an ATP regenerating system. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. ( D ) GST or GST fusion proteins were precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 19–21) or the master mix alone (lane 22) in the presence of ATP and an ATP regenerating system by using glutathione Sepharose beads. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with a rabbit polyclonal antibody directed against cdc34. The dots to the right of high molecular weight bands in lanes 6 and 17 identify ubiquitinated proteins; G, GST; 2, GST-exon 2 chimeric protein; 3, GST-carboxyl-terminal domain of exon 3 fusion protein.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin-conjugating enzyme, and possesses in vitro E3 ubiquitin ligase activity

    doi: 10.1073/pnas.161283098

    Figure Lengend Snippet: The domain encoded by the carboxyl terminus of exon 3 of ICP0 acts as an E3 ligase, whereas the sequences encoded by exon 2 bind cdc34 ubiquitin-conjugating enzyme. ( A ) Immunoblots of electrophoretically separated products of substrate-independent in vitro ubiquitination reactions. GST (lanes 1 and 2), GST-exon 2 (lanes 3 and 4), GST-exon 3 (lanes 5 and 6), and no additional protein (lane 7) were added to the substrate-independent in vitro ubiquitination reaction master mix (MM) containing recombinant Uba1 (E1), recombinant cdc34, biotinylated ubiquitin, and ubiquitination buffer in the presence and absence of ATP and an ATP regenerating system, as described in Materials and Methods . The reaction was stopped after 90 min, and the reaction mixture was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. ( B ) Electrophoretically separated reaction mixtures containing the indicated GST fusion protein in addition to the master mix (lanes 8–13) or the master mix alone (lane 14) in the presence and absence of ATP and an ATP regenerating system were probed with a rabbit polyclonal antibody directed against GST. ( C ) cdc34 was precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 15–17) or the master mix alone (lane 18) in the presence of ATP and an ATP regenerating system. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. ( D ) GST or GST fusion proteins were precipitated from reactions containing the indicated GST fusion protein in addition to the master mix (lanes 19–21) or the master mix alone (lane 22) in the presence of ATP and an ATP regenerating system by using glutathione Sepharose beads. The precipitate was electrophoretically separated in a denaturing polyacrylamide gel and probed with a rabbit polyclonal antibody directed against cdc34. The dots to the right of high molecular weight bands in lanes 6 and 17 identify ubiquitinated proteins; G, GST; 2, GST-exon 2 chimeric protein; 3, GST-carboxyl-terminal domain of exon 3 fusion protein.

    Article Snippet: Thirty microliters of in vitro reactions were performed in ubiquitination buffer [50 mM Tris, pH 7.5/2.5 mM MgCl/0.5 mM DTT] and contained 40 ng of recombinant E1 (Calbiochem catalogue no. 662070), 40 ng [His6 ]-UbcH3 (Affiniti, Mamhead, Exeter, U.K., catalogue no. UW8730), 2 μg of biotinylated ubiquitin (Affiniti catalogue no. UW8705), and 0.2 mM ATP along with the ATP regenerating system described above where indicated.

    Techniques: Western Blot, In Vitro, Recombinant, Molecular Weight

    Models for E3 ubiquitin ligase function. E1 ubiquitin-activating enzymes are shown in dark blue, E2 ubiquitin-conjugating enzymes in green, RING finger subunits and domains in yellow, substrate-binding subunits and domains in magenta, and substrates in gray. ( A ). SCF contains cullin 1 (CUL1), whereas other cullins are components of other multicomponent E3s. The cullin serves as a scaffold to bind Skp1, the RING finger protein Rbx1, and the E2 cdc34, which also interacts the RING finger. ( B ). A Src-homology 2 (SH2) domain binds phosphotyrosine, such as platelet-derived growth factor receptor β (PDGF-Rβ). ( C ) Proposed model for ICP0 unimolecular E3 ubiquitin ligase activity. Cdc34 binds the RING finger domain in ICP0-exon 2. Unidentified substrates may bind exon 3, facilitating the transfer of ubiquitin from E2 to the substrate. Also, it is possible that ICP0 E3 activity catalyzes regulatory self-ubiquitination, which does not target ICP0 for degradation.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The infected cell protein 0 of herpes simplex virus 1 dynamically interacts with proteasomes, binds and activates the cdc34 E2 ubiquitin-conjugating enzyme, and possesses in vitro E3 ubiquitin ligase activity

    doi: 10.1073/pnas.161283098

    Figure Lengend Snippet: Models for E3 ubiquitin ligase function. E1 ubiquitin-activating enzymes are shown in dark blue, E2 ubiquitin-conjugating enzymes in green, RING finger subunits and domains in yellow, substrate-binding subunits and domains in magenta, and substrates in gray. ( A ). SCF contains cullin 1 (CUL1), whereas other cullins are components of other multicomponent E3s. The cullin serves as a scaffold to bind Skp1, the RING finger protein Rbx1, and the E2 cdc34, which also interacts the RING finger. ( B ). A Src-homology 2 (SH2) domain binds phosphotyrosine, such as platelet-derived growth factor receptor β (PDGF-Rβ). ( C ) Proposed model for ICP0 unimolecular E3 ubiquitin ligase activity. Cdc34 binds the RING finger domain in ICP0-exon 2. Unidentified substrates may bind exon 3, facilitating the transfer of ubiquitin from E2 to the substrate. Also, it is possible that ICP0 E3 activity catalyzes regulatory self-ubiquitination, which does not target ICP0 for degradation.

    Article Snippet: Thirty microliters of in vitro reactions were performed in ubiquitination buffer [50 mM Tris, pH 7.5/2.5 mM MgCl/0.5 mM DTT] and contained 40 ng of recombinant E1 (Calbiochem catalogue no. 662070), 40 ng [His6 ]-UbcH3 (Affiniti, Mamhead, Exeter, U.K., catalogue no. UW8730), 2 μg of biotinylated ubiquitin (Affiniti catalogue no. UW8705), and 0.2 mM ATP along with the ATP regenerating system described above where indicated.

    Techniques: Binding Assay, Derivative Assay, Activity Assay