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Millipore anti flag m2 affinity gel
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Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Zebrafish Mbd5 binds to RNA m 5 C and regulates histone deubiquitylation and gene expression in development metabolism and behavior"

Article Title: Zebrafish Mbd5 binds to RNA m 5 C and regulates histone deubiquitylation and gene expression in development metabolism and behavior

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkae093

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Techniques Used: Clone Assay, Sequencing, Multiplex Assay, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Methylation, Software


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Millipore anti-flag
Anti Flag, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Millipore anti flag m2
Anti Flag M2, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Millipore anti flag m2 magnetic beads
Anti Flag M2 Magnetic Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Millipore anti flag m2 magnetic beads
(A) Identification of Yjr012c and C6ORF226 as uncharacterized proteins that interact with the PTS2-protein import system via BioGRID 28 and BioPlex, 29,30 respectively. A circle represents the purified protein (bait) and a diamond represents the accompanying interactor (prey). Arrows point from bait to prey. Two circles with bidirectional arrows between them indicate that both proteins have served as baits and also been identified as accompanying interactors. For S. cerevisiae , only protein-protein interactions found in at least two independent studies per BioGRID were considered. The combined BioPlex interactome from HCT116 and HEK293T cells is shown for human/ Homo sapiens ( H. sapiens ). (B) Domain analysis and alignment of Yjr012c and C6ORF226. Probabilities of disorder (determined using IUPred2A 61 ), the DUF5572, [R/K]PWE motifs, and further regions of interest are indicated. α, α-helical. (C) Sc Pex39 is a specific component of Pex18 complexes as determined by label-free quantitative affinity purification-mass spectrometry. Pex18 complexes were affinity purified from soluble fractions of oleic acid-grown wild-type and Pex18-TPA-expressing cells (n = 3). Enrichment of proteins in Pex18 complexes and Q-values were determined using the rank sum method. 62 Known peroxins and PTS2 proteins are labelled and/or marked by black dots. Dashed lines indicate a Q-value threshold of 0.05 and a fold-enrichment of 64. (D) Hs PEX39 interacts with PEX7, PHYH, ACAA1, and AGPS per assessment with human HCT116 cells. <t>Anti-FLAG</t> immunoprecipitates and cell lysates were prepared from HCT116 cells stably expressing the indicated proteins. Samples were analyzed by immunoblotting for the indicated proteins; detection of HA denoted by “ HA ” in the labelled black arrowheads identifying the corresponding proteins. Numbers along the left side indicate molecular weights (kD). Dashed lines indicate where different lanes of the same membrane were brought together. For the PHYH, ACAA1, and AGPS blots, the solid and open red arrowheads indicate the mature and precursor forms of these proteins, respectively. An asterisk indicates a non-specific band. (E) Hs PEX39 interacts with PEX7, PHYH, ACAA1, and AGPS per assessment with human HEK293T cells. Anti-FLAG immunoprecipitates and cell lysates were prepared from HEK293T cells stably expressing the indicated proteins. Samples were analyzed by immunoblotting for the indicated proteins. Occasionally, it was necessary to have blanks sandwiching a given sample to prevent spillover of immunoblot signal to other samples. Annotation of the immunoblots is otherwise the same as described for (D). (F) Hs PEX39 interacts with PEX5 per assessment with HEK293T cells. Anti-FLAG immunoprecipitates and cell lysates were prepared from HEK293T cells stably expressing the indicated proteins. Samples were analyzed by immunoblotting for the indicated proteins; detection of HA denoted by “ HA ” in the labelled black arrowheads identifying the corresponding proteins. An asterisk indicates a non-specific band. (G) Hs PEX39 can complex with PEX7, PHYH, and PEX5 in vitro . Radiolabeled H6PEX7 was pre-incubated or not with the recombinant proteins GST- Hs PEX39, H6PHYH, and H6PEX5(1-324), as indicated. Samples were analyzed by native-PAGE and autoradiography. In-gel migration of PEX7 alone (PEX7), lysate hemoglobin, and complexes PEX7- Hs PEX39 (#), PEX7-PHYH- Hs PEX39 (&), PEX7-PEX5-PHYH- Hs PEX39 ($) are indicated. The autoradiograph and the corresponding Ponceau S-stained membrane are shown. See also Figure S1.
Anti Flag M2 Magnetic Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "PEX39 facilitates the peroxisomal import of PTS2 proteins"

Article Title: PEX39 facilitates the peroxisomal import of PTS2 proteins

Journal: bioRxiv

doi: 10.1101/2024.04.30.591961

(A) Identification of Yjr012c and C6ORF226 as uncharacterized proteins that interact with the PTS2-protein import system via BioGRID 28 and BioPlex, 29,30 respectively. A circle represents the purified protein (bait) and a diamond represents the accompanying interactor (prey). Arrows point from bait to prey. Two circles with bidirectional arrows between them indicate that both proteins have served as baits and also been identified as accompanying interactors. For S. cerevisiae , only protein-protein interactions found in at least two independent studies per BioGRID were considered. The combined BioPlex interactome from HCT116 and HEK293T cells is shown for human/ Homo sapiens ( H. sapiens ). (B) Domain analysis and alignment of Yjr012c and C6ORF226. Probabilities of disorder (determined using IUPred2A 61 ), the DUF5572, [R/K]PWE motifs, and further regions of interest are indicated. α, α-helical. (C) Sc Pex39 is a specific component of Pex18 complexes as determined by label-free quantitative affinity purification-mass spectrometry. Pex18 complexes were affinity purified from soluble fractions of oleic acid-grown wild-type and Pex18-TPA-expressing cells (n = 3). Enrichment of proteins in Pex18 complexes and Q-values were determined using the rank sum method. 62 Known peroxins and PTS2 proteins are labelled and/or marked by black dots. Dashed lines indicate a Q-value threshold of 0.05 and a fold-enrichment of 64. (D) Hs PEX39 interacts with PEX7, PHYH, ACAA1, and AGPS per assessment with human HCT116 cells. Anti-FLAG immunoprecipitates and cell lysates were prepared from HCT116 cells stably expressing the indicated proteins. Samples were analyzed by immunoblotting for the indicated proteins; detection of HA denoted by “ HA ” in the labelled black arrowheads identifying the corresponding proteins. Numbers along the left side indicate molecular weights (kD). Dashed lines indicate where different lanes of the same membrane were brought together. For the PHYH, ACAA1, and AGPS blots, the solid and open red arrowheads indicate the mature and precursor forms of these proteins, respectively. An asterisk indicates a non-specific band. (E) Hs PEX39 interacts with PEX7, PHYH, ACAA1, and AGPS per assessment with human HEK293T cells. Anti-FLAG immunoprecipitates and cell lysates were prepared from HEK293T cells stably expressing the indicated proteins. Samples were analyzed by immunoblotting for the indicated proteins. Occasionally, it was necessary to have blanks sandwiching a given sample to prevent spillover of immunoblot signal to other samples. Annotation of the immunoblots is otherwise the same as described for (D). (F) Hs PEX39 interacts with PEX5 per assessment with HEK293T cells. Anti-FLAG immunoprecipitates and cell lysates were prepared from HEK293T cells stably expressing the indicated proteins. Samples were analyzed by immunoblotting for the indicated proteins; detection of HA denoted by “ HA ” in the labelled black arrowheads identifying the corresponding proteins. An asterisk indicates a non-specific band. (G) Hs PEX39 can complex with PEX7, PHYH, and PEX5 in vitro . Radiolabeled H6PEX7 was pre-incubated or not with the recombinant proteins GST- Hs PEX39, H6PHYH, and H6PEX5(1-324), as indicated. Samples were analyzed by native-PAGE and autoradiography. In-gel migration of PEX7 alone (PEX7), lysate hemoglobin, and complexes PEX7- Hs PEX39 (#), PEX7-PHYH- Hs PEX39 (&), PEX7-PEX5-PHYH- Hs PEX39 ($) are indicated. The autoradiograph and the corresponding Ponceau S-stained membrane are shown. See also Figure S1.
Figure Legend Snippet: (A) Identification of Yjr012c and C6ORF226 as uncharacterized proteins that interact with the PTS2-protein import system via BioGRID 28 and BioPlex, 29,30 respectively. A circle represents the purified protein (bait) and a diamond represents the accompanying interactor (prey). Arrows point from bait to prey. Two circles with bidirectional arrows between them indicate that both proteins have served as baits and also been identified as accompanying interactors. For S. cerevisiae , only protein-protein interactions found in at least two independent studies per BioGRID were considered. The combined BioPlex interactome from HCT116 and HEK293T cells is shown for human/ Homo sapiens ( H. sapiens ). (B) Domain analysis and alignment of Yjr012c and C6ORF226. Probabilities of disorder (determined using IUPred2A 61 ), the DUF5572, [R/K]PWE motifs, and further regions of interest are indicated. α, α-helical. (C) Sc Pex39 is a specific component of Pex18 complexes as determined by label-free quantitative affinity purification-mass spectrometry. Pex18 complexes were affinity purified from soluble fractions of oleic acid-grown wild-type and Pex18-TPA-expressing cells (n = 3). Enrichment of proteins in Pex18 complexes and Q-values were determined using the rank sum method. 62 Known peroxins and PTS2 proteins are labelled and/or marked by black dots. Dashed lines indicate a Q-value threshold of 0.05 and a fold-enrichment of 64. (D) Hs PEX39 interacts with PEX7, PHYH, ACAA1, and AGPS per assessment with human HCT116 cells. Anti-FLAG immunoprecipitates and cell lysates were prepared from HCT116 cells stably expressing the indicated proteins. Samples were analyzed by immunoblotting for the indicated proteins; detection of HA denoted by “ HA ” in the labelled black arrowheads identifying the corresponding proteins. Numbers along the left side indicate molecular weights (kD). Dashed lines indicate where different lanes of the same membrane were brought together. For the PHYH, ACAA1, and AGPS blots, the solid and open red arrowheads indicate the mature and precursor forms of these proteins, respectively. An asterisk indicates a non-specific band. (E) Hs PEX39 interacts with PEX7, PHYH, ACAA1, and AGPS per assessment with human HEK293T cells. Anti-FLAG immunoprecipitates and cell lysates were prepared from HEK293T cells stably expressing the indicated proteins. Samples were analyzed by immunoblotting for the indicated proteins. Occasionally, it was necessary to have blanks sandwiching a given sample to prevent spillover of immunoblot signal to other samples. Annotation of the immunoblots is otherwise the same as described for (D). (F) Hs PEX39 interacts with PEX5 per assessment with HEK293T cells. Anti-FLAG immunoprecipitates and cell lysates were prepared from HEK293T cells stably expressing the indicated proteins. Samples were analyzed by immunoblotting for the indicated proteins; detection of HA denoted by “ HA ” in the labelled black arrowheads identifying the corresponding proteins. An asterisk indicates a non-specific band. (G) Hs PEX39 can complex with PEX7, PHYH, and PEX5 in vitro . Radiolabeled H6PEX7 was pre-incubated or not with the recombinant proteins GST- Hs PEX39, H6PHYH, and H6PEX5(1-324), as indicated. Samples were analyzed by native-PAGE and autoradiography. In-gel migration of PEX7 alone (PEX7), lysate hemoglobin, and complexes PEX7- Hs PEX39 (#), PEX7-PHYH- Hs PEX39 (&), PEX7-PEX5-PHYH- Hs PEX39 ($) are indicated. The autoradiograph and the corresponding Ponceau S-stained membrane are shown. See also Figure S1.

Techniques Used: Purification, Affinity Purification, Mass Spectrometry, Expressing, Stable Transfection, Western Blot, Membrane, In Vitro, Incubation, Recombinant, Clear Native PAGE, Autoradiography, Migration, Staining

(A) Investigation of Hs PEX39 truncation and mutated variants using native-PAGE and radiolabeled PEX7. Depictions of the different variants are shown on the left. 35 S-H 6 PEX7 was incubated or not with the indicated recombinant proteins and analyzed by native-PAGE and autoradiography. In-gel position of PEX7 alone (PEX7), lysate hemoglobin, and of the complexes PEX7- Hs PEX39 (#), PEX7- Hs PEX39-PHYH (&), PEX7-PEX5-PHYH- Hs PEX39 ($) and PEX7-PEX5-PHYH (*) are indicated for the full-length wild-type Hs PEX39 variant. Double bands in Hs PEX39(ΔN) complexes are due to co-migration with hemoglobin from the lysate. (B) Investigation of Hs PEX39 truncation and mutated variants using an in vitro import assay. 35 S-ACAA1 was subjected to in vitro import assays at 37°C in the absence (-) or presence of the indicated recombinant Hs PEX39 proteins [see depictions of the indicated variants in left side of (A)]. After incubation, reactions were treated with trypsin and organelles were isolated by centrifugation and analyzed by SDS-PAGE and autoradiography. Precursor and mature forms of ACAA1 denoted by open and solid red arrowheads, respectively. I, 5% of the reticulocyte lysate containing the 35 S-labeled protein used in each reaction. (C) Mutation of the RPWE motif in Sc Pex39 prevents rescue of the fitness defect of Scpex39Δ cells grown on oleic acid. Experiment performed as described in analyzing the growth of Scpex39Δ cells transformed with a plasmid for expression of an Sc Pex39 RPWE-to-AAAA mutant under control of the endogenous promoter [pPEX39(4A)] in oleic acid medium. Data for wild-type, Scpex39Δ , and Scpex39Δ + pPEX39 are the same as shown in (right plot). Data are mean ± SD (n = 4). Error bars may not be visible if the SD is very small. (D) Cellular fractionation of Scpex39Δ yeast expressing wild-type or mutant Sc Pex39. Experiment performed as described in using Scpex39 Δ cells transformed with plasmids for expression of wild-type Sc Pex39 (pPEX39) or the Sc Pex39 RPWE-to-AAAA mutant [pPEX39(4A)], each under control of the endogenous promoter. PNS, post-nuclear supernatant; S, cytosolic supernatant; OP, organellar pellet. (E) Mutations of the KPWE motif of Hs PEX39 have deleterious effects on protein interactions per assessment with HEK293T cells. Anti-FLAG immunoprecipitates and cell lysates were prepared from wild-type HEK293T cells stably expressing the indicated proteins. Samples were analyzed by immunoblotting for the indicated proteins; detection of HA denoted by “ HA ” in the labeled black arrowheads identifying the corresponding proteins. GAPDH-FLAG-HA is a negative control for the immunoprecipitations. Different Hs PEX39 variants denoted as WT (wild-type) or by single or multiple alanine replacements of the indicated residue(s). (F) Close-up views of the interactions between the KPWE motif of Hs PEX39 and PEX7. Shown are different surface properties and relative sequence conservation of the Hs PEX39 binding region at the bottom face of PEX7. Images are based on the same predicted structural model as shown in ( H. sapiens ). The individual amino acids of the KPWE motif are labeled. (G) Mutation of the KPWE motif prevents overexpressed Hs PEX39 from impairing the import of PHYH, ACAA1, and AGPS in human cells. GAPDH (negative control), Hs PEX39, or Hs PEX39 with KPWE motif replaced by AAAA [ Hs PEX39(4A)] were stably overexpressed in wild-type HEK293T cells and cellular lysates analyzed by immunoblotting for the indicated proteins. For the PHYH, ACAA1, and AGPS blots, the solid and open red arrowheads indicate the mature and precursor forms of these proteins, respectively. CANX is a loading control. See also Figure S5.
Figure Legend Snippet: (A) Investigation of Hs PEX39 truncation and mutated variants using native-PAGE and radiolabeled PEX7. Depictions of the different variants are shown on the left. 35 S-H 6 PEX7 was incubated or not with the indicated recombinant proteins and analyzed by native-PAGE and autoradiography. In-gel position of PEX7 alone (PEX7), lysate hemoglobin, and of the complexes PEX7- Hs PEX39 (#), PEX7- Hs PEX39-PHYH (&), PEX7-PEX5-PHYH- Hs PEX39 ($) and PEX7-PEX5-PHYH (*) are indicated for the full-length wild-type Hs PEX39 variant. Double bands in Hs PEX39(ΔN) complexes are due to co-migration with hemoglobin from the lysate. (B) Investigation of Hs PEX39 truncation and mutated variants using an in vitro import assay. 35 S-ACAA1 was subjected to in vitro import assays at 37°C in the absence (-) or presence of the indicated recombinant Hs PEX39 proteins [see depictions of the indicated variants in left side of (A)]. After incubation, reactions were treated with trypsin and organelles were isolated by centrifugation and analyzed by SDS-PAGE and autoradiography. Precursor and mature forms of ACAA1 denoted by open and solid red arrowheads, respectively. I, 5% of the reticulocyte lysate containing the 35 S-labeled protein used in each reaction. (C) Mutation of the RPWE motif in Sc Pex39 prevents rescue of the fitness defect of Scpex39Δ cells grown on oleic acid. Experiment performed as described in analyzing the growth of Scpex39Δ cells transformed with a plasmid for expression of an Sc Pex39 RPWE-to-AAAA mutant under control of the endogenous promoter [pPEX39(4A)] in oleic acid medium. Data for wild-type, Scpex39Δ , and Scpex39Δ + pPEX39 are the same as shown in (right plot). Data are mean ± SD (n = 4). Error bars may not be visible if the SD is very small. (D) Cellular fractionation of Scpex39Δ yeast expressing wild-type or mutant Sc Pex39. Experiment performed as described in using Scpex39 Δ cells transformed with plasmids for expression of wild-type Sc Pex39 (pPEX39) or the Sc Pex39 RPWE-to-AAAA mutant [pPEX39(4A)], each under control of the endogenous promoter. PNS, post-nuclear supernatant; S, cytosolic supernatant; OP, organellar pellet. (E) Mutations of the KPWE motif of Hs PEX39 have deleterious effects on protein interactions per assessment with HEK293T cells. Anti-FLAG immunoprecipitates and cell lysates were prepared from wild-type HEK293T cells stably expressing the indicated proteins. Samples were analyzed by immunoblotting for the indicated proteins; detection of HA denoted by “ HA ” in the labeled black arrowheads identifying the corresponding proteins. GAPDH-FLAG-HA is a negative control for the immunoprecipitations. Different Hs PEX39 variants denoted as WT (wild-type) or by single or multiple alanine replacements of the indicated residue(s). (F) Close-up views of the interactions between the KPWE motif of Hs PEX39 and PEX7. Shown are different surface properties and relative sequence conservation of the Hs PEX39 binding region at the bottom face of PEX7. Images are based on the same predicted structural model as shown in ( H. sapiens ). The individual amino acids of the KPWE motif are labeled. (G) Mutation of the KPWE motif prevents overexpressed Hs PEX39 from impairing the import of PHYH, ACAA1, and AGPS in human cells. GAPDH (negative control), Hs PEX39, or Hs PEX39 with KPWE motif replaced by AAAA [ Hs PEX39(4A)] were stably overexpressed in wild-type HEK293T cells and cellular lysates analyzed by immunoblotting for the indicated proteins. For the PHYH, ACAA1, and AGPS blots, the solid and open red arrowheads indicate the mature and precursor forms of these proteins, respectively. CANX is a loading control. See also Figure S5.

Techniques Used: Clear Native PAGE, Incubation, Recombinant, Autoradiography, Variant Assay, Migration, In Vitro, Isolation, Centrifugation, SDS Page, Labeling, Mutagenesis, Transformation Assay, Plasmid Preparation, Expressing, Cell Fractionation, Stable Transfection, Western Blot, Negative Control, Residue, Sequencing, Binding Assay

(A) Structural modeling predicts that [R/K]PWE motifs of PEX39 and N-terminus of PEX13 both bind to same site of PEX7 in both human and yeast. Shown are superimpositions of the predicted models shown in and 6D. The residues of the PEX39 and PEX13 [R/K]PWE motifs of human and yeast are shown. (B) FLAG-HA-tagged Hs PEX39 does not co-immunoprecipitate Hs PEX13 per assessment with HEK293T cells. Anti-FLAG immunoprecipitates and cell lysates were prepared from HEK293T cells stably expressing the indicated proteins. Samples were analyzed by immunoblotting for the indicated proteins; detection of HA denoted by “ HA ” in the labeled black arrowheads identifying the corresponding proteins. Dashed lines indicate where different lanes of the same membrane were brought together; occasionally, it was necessary to have blanks sandwiching a given sample to prevent spillover of immunoblot signal to other samples. (C) Hs PEX39 binds to PEX7 and displaces the N-terminus of Hs PEX13 with fast kinetics in vitro . A mixture of 35 S-H6PEX7 and NtPEX13 (PEX7+NtPEX13) was incubated with a 5-fold molar excess of GST- Hs PEX39 or GST-Ub at 23°C. Aliquots before (0’) and during incubations were collected at the indicated time-points. Radiolabeled PEX7 in a mixture with GST- Hs PEX39 (PEX7+ Hs PEX39) was also analyzed. Samples were analyzed by native-PAGE and autoradiography. In-gel migration of PEX7 alone (PEX7), the complexes PEX7- Hs PEX39 (#) and PEX7-NtPEX13 (@), and other proteins are indicated. (D) Hs PEX39(ΔN) can rapidly exchange with wild-type Hs PEX39 for PEX7 binding in vitro . Mixtures of 35 S-H6PEX7 and GST- Hs PEX39 in the absence (PEX7- Hs PEX39) or presence of PHYH (PEX7-PHYH- Hs PEX39) were incubated with a 100-fold molar excess of either GST- Hs PEX39(ΔN) or GST-Ub at 23°C. Aliquots before (0’) and during incubations were collected at the indicated time-points. Samples were analyzed by native-PAGE and autoradiography. In-gel migration of PEX7 alone (PEX7), lysate hemoglobin, and the complexes PEX7- Hs PEX39 (#) and PEX7-PHYH- Hs PEX39 (&) are indicated. Double bands in PEX7- Hs PEX39(ΔN) complexes are caused by co-migration with hemoglobin from the lysate (see also ). (E) Model depicting how PEX39 facilitates PTS2-protein import and the consequences of perturbations explored in this study. Proteins and their respective motifs/domains are indicated: cargo with α (PTS2), PTS2 cargo; PEX7-BD, PEX7 binding domain; 13, PEX13; 14/17, PEX14/PEX17; blue, PTS2 co-receptor ( e.g. , PEX5/Pex18/Pex21); NTD, N-terminal domain; CTD, C-terminal domain; WxxxF, di-aromatic motif. Dashed lines highlight known protein-protein interactions. 49,51,63-66 See also Figure S7.
Figure Legend Snippet: (A) Structural modeling predicts that [R/K]PWE motifs of PEX39 and N-terminus of PEX13 both bind to same site of PEX7 in both human and yeast. Shown are superimpositions of the predicted models shown in and 6D. The residues of the PEX39 and PEX13 [R/K]PWE motifs of human and yeast are shown. (B) FLAG-HA-tagged Hs PEX39 does not co-immunoprecipitate Hs PEX13 per assessment with HEK293T cells. Anti-FLAG immunoprecipitates and cell lysates were prepared from HEK293T cells stably expressing the indicated proteins. Samples were analyzed by immunoblotting for the indicated proteins; detection of HA denoted by “ HA ” in the labeled black arrowheads identifying the corresponding proteins. Dashed lines indicate where different lanes of the same membrane were brought together; occasionally, it was necessary to have blanks sandwiching a given sample to prevent spillover of immunoblot signal to other samples. (C) Hs PEX39 binds to PEX7 and displaces the N-terminus of Hs PEX13 with fast kinetics in vitro . A mixture of 35 S-H6PEX7 and NtPEX13 (PEX7+NtPEX13) was incubated with a 5-fold molar excess of GST- Hs PEX39 or GST-Ub at 23°C. Aliquots before (0’) and during incubations were collected at the indicated time-points. Radiolabeled PEX7 in a mixture with GST- Hs PEX39 (PEX7+ Hs PEX39) was also analyzed. Samples were analyzed by native-PAGE and autoradiography. In-gel migration of PEX7 alone (PEX7), the complexes PEX7- Hs PEX39 (#) and PEX7-NtPEX13 (@), and other proteins are indicated. (D) Hs PEX39(ΔN) can rapidly exchange with wild-type Hs PEX39 for PEX7 binding in vitro . Mixtures of 35 S-H6PEX7 and GST- Hs PEX39 in the absence (PEX7- Hs PEX39) or presence of PHYH (PEX7-PHYH- Hs PEX39) were incubated with a 100-fold molar excess of either GST- Hs PEX39(ΔN) or GST-Ub at 23°C. Aliquots before (0’) and during incubations were collected at the indicated time-points. Samples were analyzed by native-PAGE and autoradiography. In-gel migration of PEX7 alone (PEX7), lysate hemoglobin, and the complexes PEX7- Hs PEX39 (#) and PEX7-PHYH- Hs PEX39 (&) are indicated. Double bands in PEX7- Hs PEX39(ΔN) complexes are caused by co-migration with hemoglobin from the lysate (see also ). (E) Model depicting how PEX39 facilitates PTS2-protein import and the consequences of perturbations explored in this study. Proteins and their respective motifs/domains are indicated: cargo with α (PTS2), PTS2 cargo; PEX7-BD, PEX7 binding domain; 13, PEX13; 14/17, PEX14/PEX17; blue, PTS2 co-receptor ( e.g. , PEX5/Pex18/Pex21); NTD, N-terminal domain; CTD, C-terminal domain; WxxxF, di-aromatic motif. Dashed lines highlight known protein-protein interactions. 49,51,63-66 See also Figure S7.

Techniques Used: Stable Transfection, Expressing, Western Blot, Labeling, Membrane, In Vitro, Incubation, Clear Native PAGE, Autoradiography, Migration, Binding Assay

monoclonal anti flag m2 peroxidase hrp antibody  (Millipore)

 
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    Millipore monoclonal anti flag m2 peroxidase hrp antibody
    Monoclonal Anti Flag M2 Peroxidase Hrp Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal anti flag m2 peroxidase hrp antibody
    Monoclonal Anti Flag M2 Peroxidase Hrp Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti flag ma4
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    Millipore anti flag m2
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    Anti Flag M2 Affinity Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti flag m2 affinity gel
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    Journal: Nucleic Acids Research

    Article Title: Zebrafish Mbd5 binds to RNA m 5 C and regulates histone deubiquitylation and gene expression in development metabolism and behavior

    doi: 10.1093/nar/gkae093

    Figure Lengend Snippet: Key resources table

    Article Snippet: The supernatants were incubated with anti-Flag M2 Affinity Gel (A2220, Millipore) or anti-IgG Agarose (A0919, Sigma-Aldrich) as a control at 4°C overnight.

    Techniques: Clone Assay, Sequencing, Multiplex Assay, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Methylation, Software