Structured Review

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
https://www.bioz.com/result/anti flag m2 magnetic beads/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti flag m2 magnetic beads - by Bioz Stars, 2024-05
86/100 stars

Images


Structured Review

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
https://www.bioz.com/result/anti flag m2 magnetic beads/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti flag m2 magnetic beads - by Bioz Stars, 2024-05
86/100 stars

Images

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


Structured Review

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
https://www.bioz.com/result/anti flag m2 magnetic beads/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti flag m2 magnetic beads - by Bioz Stars, 2024-05
86/100 stars

Images

1) Product Images from "Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity"

Article Title: Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity

Journal: Cell reports

doi: 10.1016/j.celrep.2024.113965


Figure Legend Snippet:

Techniques Used: Virus, Recombinant, Protease Inhibitor, Electron Microscopy, Lysis, Extraction, Staining, Mutagenesis, Software


Structured Review

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
https://www.bioz.com/result/anti flag m2 magnetic beads/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti flag m2 magnetic beads - by Bioz Stars, 2024-05
86/100 stars

Images


Structured Review

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
https://www.bioz.com/result/anti flag m2 magnetic beads/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti flag m2 magnetic beads - by Bioz Stars, 2024-05
86/100 stars

Images


Structured Review

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
https://www.bioz.com/result/anti flag m2 magnetic beads/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti flag m2 magnetic beads - by Bioz Stars, 2024-05
86/100 stars

Images


Structured Review

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
https://www.bioz.com/result/anti flag m2 magnetic beads/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti flag m2 magnetic beads - by Bioz Stars, 2024-05
86/100 stars

Images


Structured Review

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
https://www.bioz.com/result/anti flag m2 magnetic beads/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti flag m2 magnetic beads - by Bioz Stars, 2024-05
86/100 stars

Images


Structured Review

Millipore anti flag m2 magnetic beads
GPATCH4 binds the RNA helicase DHX15 and stimulates its ATPase activity. ( A ) Schematic views of the domain architectures of GPATCH4 (GP4) and DHX15. Domain boundaries according to UniProt ( https://www.uniprot.org/ ) are indicated with amino acid numbers given below. Amino acid substitutions predicted to impede the interaction between GPATCH4 and DHX15 are indicated in red. An amino acid substitution within motif II of the DHX15 RecA1 domain known to reduce ATPase activity is indicated in black. ( B ) Whole cell extracts from stably transfected cell lines expressing the <t>His-FLAG</t> tag, or His-FLAG-tagged GPATCH4 or derivates thereof were used for <t>anti-FLAG</t> immunoprecipitations. Input (1%) and eluate samples were analysed by western blotting using the indicated antibodies. Experiments were performed in triplicate and representative data are shown. ( C ) Whole cell extracts from stably transfected cell lines expressing the His-FLAG tag or FLAG-His-tagged DHX15 or a derivate carrying an L536E substitution were used for anti-FLAG immunoprecipitations. Input (1%) and eluate samples were analysed by western blotting using the indicated antibodies. Experiments were performed in triplicate and representative data are shown. ( D ) Recombinant MBP-DHX15-His, MBP-DHX15 E261Q -His and ZZ-GP4 GP -His purified from E. coli were separated by SDS-PAGE and visualized by Coomassie staining. ( E ) MBP-DHX15-His was incubated with ZZ-GP4 GP -His-bound IgG or unbound IgG. Input (10%) and eluates were separated by SDS-PAGE and proteins were visualized by western blotting with an anti-His antibody. Experiments were performed in triplicate and representative data are shown. ( F ) ATP hydrolysis by MBP-DHX15-His, MBP-DHX15 E261Q -His in the absence (-) and presence (+) of a model RNA substrate and/or ZZ-GP4 GP -His were monitored using an NADH-coupled ATPase assay. ATPase activity of ZZ-GP4 GP -His was also analysed. Data from three independent experiments are shown as mean ± standard deviation (error bars) and individual data points are indicated by dots. Statistical analyses were performed using one-way ANOVA ( F = 14.6, p value < 0.0001) and significance was calculated using Tukey's multiple comparisons test. The significance for selected pairs of samples is indicated as follows: **** p < 0.0001. p values for all comparisons are shown in .
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
https://www.bioz.com/result/anti flag m2 magnetic beads/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti flag m2 magnetic beads - by Bioz Stars, 2024-05
86/100 stars

Images

1) Product Images from "GPATCH4 regulates rRNA and snRNA 2′- O -methylation in both DHX15-dependent and DHX15-independent manners"

Article Title: GPATCH4 regulates rRNA and snRNA 2′- O -methylation in both DHX15-dependent and DHX15-independent manners

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkad1202

GPATCH4 binds the RNA helicase DHX15 and stimulates its ATPase activity. ( A ) Schematic views of the domain architectures of GPATCH4 (GP4) and DHX15. Domain boundaries according to UniProt ( https://www.uniprot.org/ ) are indicated with amino acid numbers given below. Amino acid substitutions predicted to impede the interaction between GPATCH4 and DHX15 are indicated in red. An amino acid substitution within motif II of the DHX15 RecA1 domain known to reduce ATPase activity is indicated in black. ( B ) Whole cell extracts from stably transfected cell lines expressing the His-FLAG tag, or His-FLAG-tagged GPATCH4 or derivates thereof were used for anti-FLAG immunoprecipitations. Input (1%) and eluate samples were analysed by western blotting using the indicated antibodies. Experiments were performed in triplicate and representative data are shown. ( C ) Whole cell extracts from stably transfected cell lines expressing the His-FLAG tag or FLAG-His-tagged DHX15 or a derivate carrying an L536E substitution were used for anti-FLAG immunoprecipitations. Input (1%) and eluate samples were analysed by western blotting using the indicated antibodies. Experiments were performed in triplicate and representative data are shown. ( D ) Recombinant MBP-DHX15-His, MBP-DHX15 E261Q -His and ZZ-GP4 GP -His purified from E. coli were separated by SDS-PAGE and visualized by Coomassie staining. ( E ) MBP-DHX15-His was incubated with ZZ-GP4 GP -His-bound IgG or unbound IgG. Input (10%) and eluates were separated by SDS-PAGE and proteins were visualized by western blotting with an anti-His antibody. Experiments were performed in triplicate and representative data are shown. ( F ) ATP hydrolysis by MBP-DHX15-His, MBP-DHX15 E261Q -His in the absence (-) and presence (+) of a model RNA substrate and/or ZZ-GP4 GP -His were monitored using an NADH-coupled ATPase assay. ATPase activity of ZZ-GP4 GP -His was also analysed. Data from three independent experiments are shown as mean ± standard deviation (error bars) and individual data points are indicated by dots. Statistical analyses were performed using one-way ANOVA ( F = 14.6, p value < 0.0001) and significance was calculated using Tukey's multiple comparisons test. The significance for selected pairs of samples is indicated as follows: **** p < 0.0001. p values for all comparisons are shown in .
Figure Legend Snippet: GPATCH4 binds the RNA helicase DHX15 and stimulates its ATPase activity. ( A ) Schematic views of the domain architectures of GPATCH4 (GP4) and DHX15. Domain boundaries according to UniProt ( https://www.uniprot.org/ ) are indicated with amino acid numbers given below. Amino acid substitutions predicted to impede the interaction between GPATCH4 and DHX15 are indicated in red. An amino acid substitution within motif II of the DHX15 RecA1 domain known to reduce ATPase activity is indicated in black. ( B ) Whole cell extracts from stably transfected cell lines expressing the His-FLAG tag, or His-FLAG-tagged GPATCH4 or derivates thereof were used for anti-FLAG immunoprecipitations. Input (1%) and eluate samples were analysed by western blotting using the indicated antibodies. Experiments were performed in triplicate and representative data are shown. ( C ) Whole cell extracts from stably transfected cell lines expressing the His-FLAG tag or FLAG-His-tagged DHX15 or a derivate carrying an L536E substitution were used for anti-FLAG immunoprecipitations. Input (1%) and eluate samples were analysed by western blotting using the indicated antibodies. Experiments were performed in triplicate and representative data are shown. ( D ) Recombinant MBP-DHX15-His, MBP-DHX15 E261Q -His and ZZ-GP4 GP -His purified from E. coli were separated by SDS-PAGE and visualized by Coomassie staining. ( E ) MBP-DHX15-His was incubated with ZZ-GP4 GP -His-bound IgG or unbound IgG. Input (10%) and eluates were separated by SDS-PAGE and proteins were visualized by western blotting with an anti-His antibody. Experiments were performed in triplicate and representative data are shown. ( F ) ATP hydrolysis by MBP-DHX15-His, MBP-DHX15 E261Q -His in the absence (-) and presence (+) of a model RNA substrate and/or ZZ-GP4 GP -His were monitored using an NADH-coupled ATPase assay. ATPase activity of ZZ-GP4 GP -His was also analysed. Data from three independent experiments are shown as mean ± standard deviation (error bars) and individual data points are indicated by dots. Statistical analyses were performed using one-way ANOVA ( F = 14.6, p value < 0.0001) and significance was calculated using Tukey's multiple comparisons test. The significance for selected pairs of samples is indicated as follows: **** p < 0.0001. p values for all comparisons are shown in .

Techniques Used: Activity Assay, Stable Transfection, Transfection, Expressing, FLAG-tag, Western Blot, Recombinant, Purification, SDS Page, Staining, Incubation, ATPase Assay, Standard Deviation

GPATCH4 and DHX15 interact in the nucleolus and co-migrate with pre-ribosomal particles. ( A ) HEK293 Flp-In cells (WT) were subjected to immunofluorescence using anti-GPATCH4 and anti-DHX15 antibodies. Nucleoli and Cajal bodies were identified using antibodies against EMG1 and coilin, respectively. Nuclear material was visualized using DAPI staining. Scale bar indicates 10 μm. Experiments were performed in triplicate and representative data are shown. ( B ) A nucleolar extract was prepared from WT cells expressing either the His-FLAG tag or GP4-His-FLAG. A cleared cell lysate (Total) was fractionated into cytosol and nuclei. Nuclei were then further fractionated into nucleoplasm and nucleoli. Samples taken throughout the fractionation procedure were analysed by western blotting using antibodies against GPATCH4 and characterized marker proteins for each cellular compartment (GAPDH - cytosol, Lamin A/C - nucleoplasm and fibrillarin – nucleoli). Experiments were performed in triplicate and representative data are shown. ( C ) Nucleolar extracts from the HEK293 cell lines expressing the His-FLAG tag or GP4-His-FLAG were used for anti-FLAG immunoprecipitation experiments. Input (1%) and eluates were analysed by western blotting using the indicated antibodies. Experiments were performed in triplicate and representative data are shown. ( D ) Total cell extracts from WT and those expressing GP4-His-FLAG were separated by sucrose density gradient centrifugation. Peaks corresponding to (pre-)ribosomal complexes are marked on an absorbance profile at 254 nm (top). Proteins present in the cell extract (In) and each fraction were subjected to western blotting using the indicated antibodies at the bottom. Marker proteins for different complexes: uS3a - (pre-)40S, UTP14A - pre-40S/90S , eL15 - (pre-)60S, and PES1 - pre-60S . Experiments were performed in triplicate and representative data are shown. The profile and blots for marker proteins are shown for the WT sample. ( E ) WT cells or those lacking GPATCH4 (GP4KO) as well as HEK293 cells treated with non-target siRNAs (siNT) or those targeting DHX15 (siDHX15) were subjected to immunofluorescence using antibodies against GPATCH4 and DHX15. Nucleoli were identified using antibodies against EMG1. Nuclear material was visualized using DAPI staining. Scale bar indicates 10 μm. Experiments were performed in triplicate and representative data are shown. ( F ) Total cell extracts from cells as in ( E ) were separated be sucrose density gradient centrifugation and analysed as in (D). Experiments were performed in duplicate and representative data are shown.
Figure Legend Snippet: GPATCH4 and DHX15 interact in the nucleolus and co-migrate with pre-ribosomal particles. ( A ) HEK293 Flp-In cells (WT) were subjected to immunofluorescence using anti-GPATCH4 and anti-DHX15 antibodies. Nucleoli and Cajal bodies were identified using antibodies against EMG1 and coilin, respectively. Nuclear material was visualized using DAPI staining. Scale bar indicates 10 μm. Experiments were performed in triplicate and representative data are shown. ( B ) A nucleolar extract was prepared from WT cells expressing either the His-FLAG tag or GP4-His-FLAG. A cleared cell lysate (Total) was fractionated into cytosol and nuclei. Nuclei were then further fractionated into nucleoplasm and nucleoli. Samples taken throughout the fractionation procedure were analysed by western blotting using antibodies against GPATCH4 and characterized marker proteins for each cellular compartment (GAPDH - cytosol, Lamin A/C - nucleoplasm and fibrillarin – nucleoli). Experiments were performed in triplicate and representative data are shown. ( C ) Nucleolar extracts from the HEK293 cell lines expressing the His-FLAG tag or GP4-His-FLAG were used for anti-FLAG immunoprecipitation experiments. Input (1%) and eluates were analysed by western blotting using the indicated antibodies. Experiments were performed in triplicate and representative data are shown. ( D ) Total cell extracts from WT and those expressing GP4-His-FLAG were separated by sucrose density gradient centrifugation. Peaks corresponding to (pre-)ribosomal complexes are marked on an absorbance profile at 254 nm (top). Proteins present in the cell extract (In) and each fraction were subjected to western blotting using the indicated antibodies at the bottom. Marker proteins for different complexes: uS3a - (pre-)40S, UTP14A - pre-40S/90S , eL15 - (pre-)60S, and PES1 - pre-60S . Experiments were performed in triplicate and representative data are shown. The profile and blots for marker proteins are shown for the WT sample. ( E ) WT cells or those lacking GPATCH4 (GP4KO) as well as HEK293 cells treated with non-target siRNAs (siNT) or those targeting DHX15 (siDHX15) were subjected to immunofluorescence using antibodies against GPATCH4 and DHX15. Nucleoli were identified using antibodies against EMG1. Nuclear material was visualized using DAPI staining. Scale bar indicates 10 μm. Experiments were performed in triplicate and representative data are shown. ( F ) Total cell extracts from cells as in ( E ) were separated be sucrose density gradient centrifugation and analysed as in (D). Experiments were performed in duplicate and representative data are shown.

Techniques Used: Immunofluorescence, Staining, Expressing, FLAG-tag, Fractionation, Western Blot, Marker, Immunoprecipitation, Gradient Centrifugation

GPATCH4 associates with rDNA and pre-rRNA. ( A ) HEK293 cell lines expressing the His-FLAG tag, GP4-HisFLAG or, as a positive control NOLC1-His-FLAG, were crosslinked using EGS and PFA and subjected to chromatin immunoprecipitation (ChIP) via the FLAG tag. Sequencing reads were mapped to the rDNA locus and coverage tracks (scale for all tracks, 0–20 RPM) are shown above a schematic view of the rDNA locus. Two independent experiments were performed and data from one replicate (paired-end sequencing) are shown. ( B ) HEK293 cell lines expressing the His-FLAG tag or GP4-His-FLAG were UV crosslinked and RNA-protein complexes were enriched by anti-FLAG immunoprecipitation. Co-purified RNAs were trimmed, 5′ end labelled with [ 32 P] and ligated to sequencing adaptors. Complexes were separated by denaturing PAGE, transferred to nitrocellulose membrane and labelled RNAs were detected by autoradiography. Boxes indicate the areas of the membrane excised for further analysis. Two independent experiments were performed and representative data are shown. ( C ) Sequencing reads were mapped to the human genome and significant peaks present in the GP4-His-FLAG_1 and GP4-His-FLAG_2 samples that did not overlap with significant peaks in the FLAG sample were used for further analyses. Shown are the relative numbers of reads derived from different types of RNA. Abbreviations: miRNA – microRNA; rRNA – (pre-)ribosomal RNA; snoRNA – small nucleolar RNA; tRNA – transfer RNA. ( D ) Peaks on the rDNA region encoding the 47S pre-rRNA that are significant in the two GP4-His-FLAG datasets compared to the two His-FLAG datasets are shown above a schematic view of the pre-rRNA transcript. Mature rRNA sequences are shown as rectangle, and external transcribed spacers (5′ ETS and 3′ ETS) and internal transcribed spacers (ITS1 and ITS2) are depicted as black lines.
Figure Legend Snippet: GPATCH4 associates with rDNA and pre-rRNA. ( A ) HEK293 cell lines expressing the His-FLAG tag, GP4-HisFLAG or, as a positive control NOLC1-His-FLAG, were crosslinked using EGS and PFA and subjected to chromatin immunoprecipitation (ChIP) via the FLAG tag. Sequencing reads were mapped to the rDNA locus and coverage tracks (scale for all tracks, 0–20 RPM) are shown above a schematic view of the rDNA locus. Two independent experiments were performed and data from one replicate (paired-end sequencing) are shown. ( B ) HEK293 cell lines expressing the His-FLAG tag or GP4-His-FLAG were UV crosslinked and RNA-protein complexes were enriched by anti-FLAG immunoprecipitation. Co-purified RNAs were trimmed, 5′ end labelled with [ 32 P] and ligated to sequencing adaptors. Complexes were separated by denaturing PAGE, transferred to nitrocellulose membrane and labelled RNAs were detected by autoradiography. Boxes indicate the areas of the membrane excised for further analysis. Two independent experiments were performed and representative data are shown. ( C ) Sequencing reads were mapped to the human genome and significant peaks present in the GP4-His-FLAG_1 and GP4-His-FLAG_2 samples that did not overlap with significant peaks in the FLAG sample were used for further analyses. Shown are the relative numbers of reads derived from different types of RNA. Abbreviations: miRNA – microRNA; rRNA – (pre-)ribosomal RNA; snoRNA – small nucleolar RNA; tRNA – transfer RNA. ( D ) Peaks on the rDNA region encoding the 47S pre-rRNA that are significant in the two GP4-His-FLAG datasets compared to the two His-FLAG datasets are shown above a schematic view of the pre-rRNA transcript. Mature rRNA sequences are shown as rectangle, and external transcribed spacers (5′ ETS and 3′ ETS) and internal transcribed spacers (ITS1 and ITS2) are depicted as black lines.

Techniques Used: Expressing, FLAG-tag, Positive Control, Chromatin Immunoprecipitation, Sequencing, Immunoprecipitation, Purification, Membrane, Autoradiography, Derivative Assay

Components of the snoRNP machinery are bound by GPATCH4. ( A ) Extracts from HEK293 cells expressing the His-FLAG tag or GP4-His-FLAG were used for anti-FLAG immunoprecipitation experiments. RNAs extracted from the eluates were subjected to small RNA-seq. Two independent experiments were performed and averaged data are shown. The relative proportion of sequencing reads mapping to different types of small RNAs is shown. Abbreviations: snoRNA – small nucleolar RNA; scaRNA – small Cajal body-associated RNA. ( B ) The distribution of sequencing reads derived from different types of snoRNAs in the samples described in (A) is shown. ( C ) HEK293 cells expressing the His-FLAG tag or GP4-His-FLAG were UV crosslinked and whole cell extracts were used for anti-FLAG immunoprecipitation. RNAs present in inputs (0.5%) and eluates were separated by denaturing polyacrylamide gel electrophoresis and analysed by northern blotting to detect the indicated snoRNAs/scaRNAs, and tRNA Met(CAU) served as a negative control. Experiments were performed in triplicate and representative data are shown. ( D ) HEK293 cell expressing the His-FLAG tag or GP4-His-FLAG were UV crosslinked (254 nm) or cells were grown in the presence of 4-thiouridine and then crosslinked using light at 365 nm. Cleared extracts were then used for anti-FLAG immunoprecipitation. Eluates were protease- and RNase-treated and protein-RNA crosslinked peptides were identified by mass spectrometry. Amino acids of GPATCH4 crosslinked to nucleotides are indicated on a scheme of GPATCH4 with the domain architecture marked as in Figure . ( E ) Whole cell extracts from HEK293 cells expressing the His-FLAG tag or GP4-His-FLAG were used for anti-FLAG immunoprecipitation in the absence (–) and presence (+) of RNase A. Input (1%) and eluate samples were analysed by western blotting using antibodies against the snoRNP-associated proteins indicated. Experiments were performed in duplicate and representative data are shown.
Figure Legend Snippet: Components of the snoRNP machinery are bound by GPATCH4. ( A ) Extracts from HEK293 cells expressing the His-FLAG tag or GP4-His-FLAG were used for anti-FLAG immunoprecipitation experiments. RNAs extracted from the eluates were subjected to small RNA-seq. Two independent experiments were performed and averaged data are shown. The relative proportion of sequencing reads mapping to different types of small RNAs is shown. Abbreviations: snoRNA – small nucleolar RNA; scaRNA – small Cajal body-associated RNA. ( B ) The distribution of sequencing reads derived from different types of snoRNAs in the samples described in (A) is shown. ( C ) HEK293 cells expressing the His-FLAG tag or GP4-His-FLAG were UV crosslinked and whole cell extracts were used for anti-FLAG immunoprecipitation. RNAs present in inputs (0.5%) and eluates were separated by denaturing polyacrylamide gel electrophoresis and analysed by northern blotting to detect the indicated snoRNAs/scaRNAs, and tRNA Met(CAU) served as a negative control. Experiments were performed in triplicate and representative data are shown. ( D ) HEK293 cell expressing the His-FLAG tag or GP4-His-FLAG were UV crosslinked (254 nm) or cells were grown in the presence of 4-thiouridine and then crosslinked using light at 365 nm. Cleared extracts were then used for anti-FLAG immunoprecipitation. Eluates were protease- and RNase-treated and protein-RNA crosslinked peptides were identified by mass spectrometry. Amino acids of GPATCH4 crosslinked to nucleotides are indicated on a scheme of GPATCH4 with the domain architecture marked as in Figure . ( E ) Whole cell extracts from HEK293 cells expressing the His-FLAG tag or GP4-His-FLAG were used for anti-FLAG immunoprecipitation in the absence (–) and presence (+) of RNase A. Input (1%) and eluate samples were analysed by western blotting using antibodies against the snoRNP-associated proteins indicated. Experiments were performed in duplicate and representative data are shown.

Techniques Used: Expressing, FLAG-tag, Immunoprecipitation, RNA Sequencing Assay, Sequencing, Derivative Assay, Polyacrylamide Gel Electrophoresis, Northern Blot, Negative Control, Mass Spectrometry, Western Blot


Structured Review

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
https://www.bioz.com/result/anti flag m2 magnetic beads/product/Millipore
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti flag m2 magnetic beads - by Bioz Stars, 2024-05
86/100 stars

Images

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    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
    https://www.bioz.com/result/anti flag m2 magnetic beads/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti flag m2 magnetic beads - by Bioz Stars, 2024-05
    86/100 stars
      Buy from Supplier

    Image Search Results