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Bio-Rad affigel 10 slurry resin
Src kinase signaling is mediated by TLR4–Src interaction. Membranous protein fractions were immunoprecipitated by <t>Affigel</t> 10 resin–TLR4 antibody conjugate and analyzed by Western blot. a p-TLR4 level expressed as a ratio of p-TLR4 to that
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Images

1) Product Images from "Alcohol-Induced Interactive Phosphorylation of Src and Toll-like Receptor Regulates the Secretion of Inflammatory Mediators by Human Astrocytes"

Article Title: Alcohol-Induced Interactive Phosphorylation of Src and Toll-like Receptor Regulates the Secretion of Inflammatory Mediators by Human Astrocytes

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

doi: 10.1007/s11481-010-9213-z

Src kinase signaling is mediated by TLR4–Src interaction. Membranous protein fractions were immunoprecipitated by Affigel 10 resin–TLR4 antibody conjugate and analyzed by Western blot. a p-TLR4 level expressed as a ratio of p-TLR4 to that
Figure Legend Snippet: Src kinase signaling is mediated by TLR4–Src interaction. Membranous protein fractions were immunoprecipitated by Affigel 10 resin–TLR4 antibody conjugate and analyzed by Western blot. a p-TLR4 level expressed as a ratio of p-TLR4 to that

Techniques Used: Immunoprecipitation, Western Blot

Activation of cPLA2 is mediated by Src kinase signaling. Cytosolic protein fractions were immunoprecipitated by Affigel 10 resin–cPLA2 antibody conjugate and analyzed by Western blot for the expression of p-cPLA2, cPLA2, and p-Src protein. a phosphorylated
Figure Legend Snippet: Activation of cPLA2 is mediated by Src kinase signaling. Cytosolic protein fractions were immunoprecipitated by Affigel 10 resin–cPLA2 antibody conjugate and analyzed by Western blot for the expression of p-cPLA2, cPLA2, and p-Src protein. a phosphorylated

Techniques Used: Activation Assay, Immunoprecipitation, Western Blot, Expressing

2) Product Images from "Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules"

Article Title: Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules

Journal: Biochemical Journal

doi: 10.1042/BJ20040622

The last six C-terminal amino acids and the N-myristoyl moiety are required for the binding of p22 to GAPDH ( A ) Upper panel: Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of myr-p22-Δ190-195 (lanes 4–6). Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or myr-p22-Δ190-195 (lanes 3). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 or myr-p22-Δ190-195 (0.01 μg each) was used as the control. ( B ) Upper panel: Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or p22-rec (lanes 3). Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of p22-rec (lanes 4–6). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 (0.05 μg) or p22-rec (0.01 μg) was used as the control. ( A , B ) Lower panels: quantification of relative percentage of myr-p22, myr-p22-Δ190-195 or p22-rec binding to GAPDH- or ovalbumin-beads. In ( A , B ) lanes 1–6 in upper panels correspond to lanes 1–6 in lower panels. In ( A , B ) lower panels, the amount of myr-p22 associated with GAPDH-coupled beads when 4 μg of myr-p22 was incubated with 50 μg of GAPDH is considered as 100%.
Figure Legend Snippet: The last six C-terminal amino acids and the N-myristoyl moiety are required for the binding of p22 to GAPDH ( A ) Upper panel: Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of myr-p22-Δ190-195 (lanes 4–6). Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or myr-p22-Δ190-195 (lanes 3). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 or myr-p22-Δ190-195 (0.01 μg each) was used as the control. ( B ) Upper panel: Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or p22-rec (lanes 3). Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of p22-rec (lanes 4–6). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 (0.05 μg) or p22-rec (0.01 μg) was used as the control. ( A , B ) Lower panels: quantification of relative percentage of myr-p22, myr-p22-Δ190-195 or p22-rec binding to GAPDH- or ovalbumin-beads. In ( A , B ) lanes 1–6 in upper panels correspond to lanes 1–6 in lower panels. In ( A , B ) lower panels, the amount of myr-p22 associated with GAPDH-coupled beads when 4 μg of myr-p22 was incubated with 50 μg of GAPDH is considered as 100%.

Techniques Used: Binding Assay, Incubation

p22 interacts directly with GAPDH in vitro ( A ) Blot overlays were performed on 5 μg of purified rabbit muscle GAPDH, BSA or ovalbumin (Ovalb.) in the presence (+) or absence (–) of myr-p22. ( B ) Affigel-10 beads coupled with GAPDH, BSA or aldolase were incubated in the presence (+) or absence (–) of myr-p22. The amount of myr-p22 bound to the beads was detected by immunoblotting using anti-p22. ( C ) K D analysis of the binding of myr-p22 to GAPDH was performed by incubating the indicated concentrations of myr-p22 (molar amounts) with 50 μg of GAPDH-coupled with beads. The amount of bound myr-p22 was detected by immunoblotting with anti-p22. The values for K D and B max were obtained from three repeat experiments as described in the Experimental section.
Figure Legend Snippet: p22 interacts directly with GAPDH in vitro ( A ) Blot overlays were performed on 5 μg of purified rabbit muscle GAPDH, BSA or ovalbumin (Ovalb.) in the presence (+) or absence (–) of myr-p22. ( B ) Affigel-10 beads coupled with GAPDH, BSA or aldolase were incubated in the presence (+) or absence (–) of myr-p22. The amount of myr-p22 bound to the beads was detected by immunoblotting using anti-p22. ( C ) K D analysis of the binding of myr-p22 to GAPDH was performed by incubating the indicated concentrations of myr-p22 (molar amounts) with 50 μg of GAPDH-coupled with beads. The amount of bound myr-p22 was detected by immunoblotting with anti-p22. The values for K D and B max were obtained from three repeat experiments as described in the Experimental section.

Techniques Used: In Vitro, Purification, Incubation, Binding Assay

3) Product Images from "Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules"

Article Title: Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules

Journal: Biochemical Journal

doi: 10.1042/BJ20040622

The last six C-terminal amino acids and the N-myristoyl moiety are required for the binding of p22 to GAPDH ( A ) Upper panel: Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of myr-p22-Δ190-195 (lanes 4–6). Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or myr-p22-Δ190-195 (lanes 3). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 or myr-p22-Δ190-195 (0.01 μg each) was used as the control. ( B ) Upper panel: Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or p22-rec (lanes 3). Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of p22-rec (lanes 4–6). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 (0.05 μg) or p22-rec (0.01 μg) was used as the control. ( A , B ) Lower panels: quantification of relative percentage of myr-p22, myr-p22-Δ190-195 or p22-rec binding to GAPDH- or ovalbumin-beads. In ( A , B ) lanes 1–6 in upper panels correspond to lanes 1–6 in lower panels. In ( A , B ) lower panels, the amount of myr-p22 associated with GAPDH-coupled beads when 4 μg of myr-p22 was incubated with 50 μg of GAPDH is considered as 100%.
Figure Legend Snippet: The last six C-terminal amino acids and the N-myristoyl moiety are required for the binding of p22 to GAPDH ( A ) Upper panel: Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of myr-p22-Δ190-195 (lanes 4–6). Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or myr-p22-Δ190-195 (lanes 3). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 or myr-p22-Δ190-195 (0.01 μg each) was used as the control. ( B ) Upper panel: Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or p22-rec (lanes 3). Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of p22-rec (lanes 4–6). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 (0.05 μg) or p22-rec (0.01 μg) was used as the control. ( A , B ) Lower panels: quantification of relative percentage of myr-p22, myr-p22-Δ190-195 or p22-rec binding to GAPDH- or ovalbumin-beads. In ( A , B ) lanes 1–6 in upper panels correspond to lanes 1–6 in lower panels. In ( A , B ) lower panels, the amount of myr-p22 associated with GAPDH-coupled beads when 4 μg of myr-p22 was incubated with 50 μg of GAPDH is considered as 100%.

Techniques Used: Binding Assay, Incubation

p22 interacts directly with GAPDH in vitro ( A ) Blot overlays were performed on 5 μg of purified rabbit muscle GAPDH, BSA or ovalbumin (Ovalb.) in the presence (+) or absence (–) of myr-p22. ( B ) Affigel-10 beads coupled with GAPDH, BSA or aldolase were incubated in the presence (+) or absence (–) of myr-p22. The amount of myr-p22 bound to the beads was detected by immunoblotting using anti-p22. ( C ) K D analysis of the binding of myr-p22 to GAPDH was performed by incubating the indicated concentrations of myr-p22 (molar amounts) with 50 μg of GAPDH-coupled with beads. The amount of bound myr-p22 was detected by immunoblotting with anti-p22. The values for K D and B max were obtained from three repeat experiments as described in the Experimental section.
Figure Legend Snippet: p22 interacts directly with GAPDH in vitro ( A ) Blot overlays were performed on 5 μg of purified rabbit muscle GAPDH, BSA or ovalbumin (Ovalb.) in the presence (+) or absence (–) of myr-p22. ( B ) Affigel-10 beads coupled with GAPDH, BSA or aldolase were incubated in the presence (+) or absence (–) of myr-p22. The amount of myr-p22 bound to the beads was detected by immunoblotting using anti-p22. ( C ) K D analysis of the binding of myr-p22 to GAPDH was performed by incubating the indicated concentrations of myr-p22 (molar amounts) with 50 μg of GAPDH-coupled with beads. The amount of bound myr-p22 was detected by immunoblotting with anti-p22. The values for K D and B max were obtained from three repeat experiments as described in the Experimental section.

Techniques Used: In Vitro, Purification, Incubation, Binding Assay

4) Product Images from "Yra1 Is Required for S Phase Entry and Affects Dia2 Binding to Replication Origins ▿"

Article Title: Yra1 Is Required for S Phase Entry and Affects Dia2 Binding to Replication Origins ▿

Journal:

doi: 10.1128/MCB.02052-06

Dia2 binds Yra1. (A) Yra1 binds a GST-Dia2 affinity column and elutes at 0.5 M KCl. Recombinant GST-Dia2 was purified from E. coli and coupled to Affigel-10 to generate an affinity column. An extract from dia2 Δ cells was passed over the affinity
Figure Legend Snippet: Dia2 binds Yra1. (A) Yra1 binds a GST-Dia2 affinity column and elutes at 0.5 M KCl. Recombinant GST-Dia2 was purified from E. coli and coupled to Affigel-10 to generate an affinity column. An extract from dia2 Δ cells was passed over the affinity

Techniques Used: Affinity Column, Recombinant, Purification

5) Product Images from "Prefoldin-Nascent Chain Complexes in the Folding of Cytoskeletal Proteins "

Article Title: Prefoldin-Nascent Chain Complexes in the Folding of Cytoskeletal Proteins

Journal: The Journal of Cell Biology

doi:

Actin species I contains PFD. (A) HeLa cell lysate or rabbit reticulocyte lysate was examined for content of PFD 6 by SDS-PAGE and Western blot analysis. Lane 1, HeLa cells grown at 37°C; lane 2, HeLa cells 12 h after a 43°C/60 min heat shock treatment; lane 3, rabbit reticulocyte lysate. The position of PFD 6 is shown on the right of the panel. (B) Purified bovine PFD (lane 1) and in vitro translated [ 35 S]methionine-labeled actin (lane 2) were analyzed by native-PAGE, the proteins transferred to nitrocellulose, and the position of purified PFD was determined by immunoblot using the PFD 6 antibody. After extensive washing, the nitrocellulose was placed on film and the position of actin species I and II revealed by autoradiography. The positions of actin species I and II are indicated on the right. (C) Identification of actin-containing complex components by electrophoretic mobility shift assays. Full-length actin mRNA (left) was translated for 15 and 50 min. The reactions were mixed together to create a pool of all actin-containing species. The mRNA encoding 336–amino acid actin was translated for 30 min (right). Before native-PAGE analysis, equal aliquots of the reaction mixtures were incubated with: PBS (control, lane 1); preimmune antiserum (lane 2); anti–PFD 6 serum (lane 3); anti–PFD 6 serum supplemented with purified actin (lane 4); purified anti-CCT mAb (lane 5); and DNase I (lane 6). DNase shift (lane 6) was omitted for the 336–amino acid actin translation products. The different protein complexes were then analyzed by native-PAGE. A fluorogram of the gel is shown. Molecular mass markers are shown at the left, and the positions of the different actin complexes are indicated in the center. The arrowhead indicates the shift in full-length actin migration due to the presence of actin-binding proteins present in the crude rabbit antisera. (D and F) Identification of actin-containing complex components by immunodepletion with immobilized antichaperone specific antibodies and DNase I. The full-length [ 35 S]methionine-labeled actin translation reaction products (C) were incubated with antichaperone antibodies first bound to protein A–Sepharose, or with DNase I coupled to Affigel-10. After incubation, the samples were clarified and the corresponding supernatants analyzed for the presence of the different actin species by native-PAGE as shown in D. In parallel, the corresponding pellets containing the immobilized antibodies or DNase I were resuspended in Laemmli sample buffer and analyzed for their relative content of radiolabeled actin by SDS-PAGE as shown in F. An aliquot of the in vitro translation products (i.e., starting material) is shown in lane 1; protein A–Sepharose, lane 2; immobilized preimmune antibodies, lane 3; PFD 6 antibody, lane 4; anti-CCT antibody, lane 5; immobilized DNase, lane 6. The positions of the different actin complexes are indicated in the center. (E and G) The [ 35 S]methionine-labeled 336–amino acid actin translation reaction products (C) were incubated with the immobilized antichaperone antibodies or immobilized DNase. Subsequently, the samples were clarified and the supernatants and pellets analyzed by native-PAGE and SDS-PAGE, respectively. Lane designations are the same as in D and F.
Figure Legend Snippet: Actin species I contains PFD. (A) HeLa cell lysate or rabbit reticulocyte lysate was examined for content of PFD 6 by SDS-PAGE and Western blot analysis. Lane 1, HeLa cells grown at 37°C; lane 2, HeLa cells 12 h after a 43°C/60 min heat shock treatment; lane 3, rabbit reticulocyte lysate. The position of PFD 6 is shown on the right of the panel. (B) Purified bovine PFD (lane 1) and in vitro translated [ 35 S]methionine-labeled actin (lane 2) were analyzed by native-PAGE, the proteins transferred to nitrocellulose, and the position of purified PFD was determined by immunoblot using the PFD 6 antibody. After extensive washing, the nitrocellulose was placed on film and the position of actin species I and II revealed by autoradiography. The positions of actin species I and II are indicated on the right. (C) Identification of actin-containing complex components by electrophoretic mobility shift assays. Full-length actin mRNA (left) was translated for 15 and 50 min. The reactions were mixed together to create a pool of all actin-containing species. The mRNA encoding 336–amino acid actin was translated for 30 min (right). Before native-PAGE analysis, equal aliquots of the reaction mixtures were incubated with: PBS (control, lane 1); preimmune antiserum (lane 2); anti–PFD 6 serum (lane 3); anti–PFD 6 serum supplemented with purified actin (lane 4); purified anti-CCT mAb (lane 5); and DNase I (lane 6). DNase shift (lane 6) was omitted for the 336–amino acid actin translation products. The different protein complexes were then analyzed by native-PAGE. A fluorogram of the gel is shown. Molecular mass markers are shown at the left, and the positions of the different actin complexes are indicated in the center. The arrowhead indicates the shift in full-length actin migration due to the presence of actin-binding proteins present in the crude rabbit antisera. (D and F) Identification of actin-containing complex components by immunodepletion with immobilized antichaperone specific antibodies and DNase I. The full-length [ 35 S]methionine-labeled actin translation reaction products (C) were incubated with antichaperone antibodies first bound to protein A–Sepharose, or with DNase I coupled to Affigel-10. After incubation, the samples were clarified and the corresponding supernatants analyzed for the presence of the different actin species by native-PAGE as shown in D. In parallel, the corresponding pellets containing the immobilized antibodies or DNase I were resuspended in Laemmli sample buffer and analyzed for their relative content of radiolabeled actin by SDS-PAGE as shown in F. An aliquot of the in vitro translation products (i.e., starting material) is shown in lane 1; protein A–Sepharose, lane 2; immobilized preimmune antibodies, lane 3; PFD 6 antibody, lane 4; anti-CCT antibody, lane 5; immobilized DNase, lane 6. The positions of the different actin complexes are indicated in the center. (E and G) The [ 35 S]methionine-labeled 336–amino acid actin translation reaction products (C) were incubated with the immobilized antichaperone antibodies or immobilized DNase. Subsequently, the samples were clarified and the supernatants and pellets analyzed by native-PAGE and SDS-PAGE, respectively. Lane designations are the same as in D and F.

Techniques Used: SDS Page, Western Blot, Purification, In Vitro, Labeling, Clear Native PAGE, Autoradiography, Electrophoretic Mobility Shift Assay, Incubation, Migration, Binding Assay

6) Product Images from "MISTIC-fusion proteins as antigens for high quality membrane protein antibodies"

Article Title: MISTIC-fusion proteins as antigens for high quality membrane protein antibodies

Journal: Scientific Reports

doi: 10.1038/srep41519

Antisera raised against MISTIC-fusion proteins can be efficiently affinity purified. An antiserum raised against a MISTIC-fusion with the full-length transmembrane nucleoporin POM121 was affinity purified using the MISTIC-POM121 protein coupled to Affigel 10 matrix as bait. The unpurified antiserum (at a 1:1,000 dilution) as well as the affinity purified antibodies (at a 0.1 mg/ml and 0.03 mg/ml dilution) were tested by western blotting after 6% SDS-PAGE of 10 μg, 3 μg and 1 μg of a total membrane fraction from Xenopus egg extracts. Molecular size markers as well as the position of POM121 are indicated.
Figure Legend Snippet: Antisera raised against MISTIC-fusion proteins can be efficiently affinity purified. An antiserum raised against a MISTIC-fusion with the full-length transmembrane nucleoporin POM121 was affinity purified using the MISTIC-POM121 protein coupled to Affigel 10 matrix as bait. The unpurified antiserum (at a 1:1,000 dilution) as well as the affinity purified antibodies (at a 0.1 mg/ml and 0.03 mg/ml dilution) were tested by western blotting after 6% SDS-PAGE of 10 μg, 3 μg and 1 μg of a total membrane fraction from Xenopus egg extracts. Molecular size markers as well as the position of POM121 are indicated.

Techniques Used: Affinity Purification, Western Blot, SDS Page

7) Product Images from "Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules"

Article Title: Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules

Journal: Biochemical Journal

doi: 10.1042/BJ20040622

The last six C-terminal amino acids and the N-myristoyl moiety are required for the binding of p22 to GAPDH ( A ) Upper panel: Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of myr-p22-Δ190-195 (lanes 4–6). Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or myr-p22-Δ190-195 (lanes 3). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 or myr-p22-Δ190-195 (0.01 μg each) was used as the control. ( B ) Upper panel: Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or p22-rec (lanes 3). Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of p22-rec (lanes 4–6). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 (0.05 μg) or p22-rec (0.01 μg) was used as the control. ( A , B ) Lower panels: quantification of relative percentage of myr-p22, myr-p22-Δ190-195 or p22-rec binding to GAPDH- or ovalbumin-beads. In ( A , B ) lanes 1–6 in upper panels correspond to lanes 1–6 in lower panels. In ( A , B ) lower panels, the amount of myr-p22 associated with GAPDH-coupled beads when 4 μg of myr-p22 was incubated with 50 μg of GAPDH is considered as 100%.
Figure Legend Snippet: The last six C-terminal amino acids and the N-myristoyl moiety are required for the binding of p22 to GAPDH ( A ) Upper panel: Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of myr-p22-Δ190-195 (lanes 4–6). Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or myr-p22-Δ190-195 (lanes 3). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 or myr-p22-Δ190-195 (0.01 μg each) was used as the control. ( B ) Upper panel: Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or p22-rec (lanes 3). Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of p22-rec (lanes 4–6). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 (0.05 μg) or p22-rec (0.01 μg) was used as the control. ( A , B ) Lower panels: quantification of relative percentage of myr-p22, myr-p22-Δ190-195 or p22-rec binding to GAPDH- or ovalbumin-beads. In ( A , B ) lanes 1–6 in upper panels correspond to lanes 1–6 in lower panels. In ( A , B ) lower panels, the amount of myr-p22 associated with GAPDH-coupled beads when 4 μg of myr-p22 was incubated with 50 μg of GAPDH is considered as 100%.

Techniques Used: Binding Assay, Incubation

p22 interacts directly with GAPDH in vitro ( A ) Blot overlays were performed on 5 μg of purified rabbit muscle GAPDH, BSA or ovalbumin (Ovalb.) in the presence (+) or absence (–) of myr-p22. ( B ) Affigel-10 beads coupled with GAPDH, BSA or aldolase were incubated in the presence (+) or absence (–) of myr-p22. The amount of myr-p22 bound to the beads was detected by immunoblotting using anti-p22. ( C ) K D analysis of the binding of myr-p22 to GAPDH was performed by incubating the indicated concentrations of myr-p22 (molar amounts) with 50 μg of GAPDH-coupled with beads. The amount of bound myr-p22 was detected by immunoblotting with anti-p22. The values for K D and B max were obtained from three repeat experiments as described in the Experimental section.
Figure Legend Snippet: p22 interacts directly with GAPDH in vitro ( A ) Blot overlays were performed on 5 μg of purified rabbit muscle GAPDH, BSA or ovalbumin (Ovalb.) in the presence (+) or absence (–) of myr-p22. ( B ) Affigel-10 beads coupled with GAPDH, BSA or aldolase were incubated in the presence (+) or absence (–) of myr-p22. The amount of myr-p22 bound to the beads was detected by immunoblotting using anti-p22. ( C ) K D analysis of the binding of myr-p22 to GAPDH was performed by incubating the indicated concentrations of myr-p22 (molar amounts) with 50 μg of GAPDH-coupled with beads. The amount of bound myr-p22 was detected by immunoblotting with anti-p22. The values for K D and B max were obtained from three repeat experiments as described in the Experimental section.

Techniques Used: In Vitro, Purification, Incubation, Binding Assay

8) Product Images from "Engineering streptokinase for generation of active site-labeled plasminogen analogs *"

Article Title: Engineering streptokinase for generation of active site-labeled plasminogen analogs *

Journal: Analytical biochemistry

doi: 10.1016/j.ab.2011.04.025

Purification of labeled [Lys]Pg from labeling reaction mixtures by Ni 2+ -iminodiacetic acid-Sepharose and SK-AffiGel-10 affinity chromatography
Figure Legend Snippet: Purification of labeled [Lys]Pg from labeling reaction mixtures by Ni 2+ -iminodiacetic acid-Sepharose and SK-AffiGel-10 affinity chromatography

Techniques Used: Purification, Labeling, Affinity Chromatography

9) Product Images from "MISTIC-fusion proteins as antigens for high quality membrane protein antibodies"

Article Title: MISTIC-fusion proteins as antigens for high quality membrane protein antibodies

Journal: Scientific Reports

doi: 10.1038/srep41519

Antisera raised against MISTIC-fusion proteins can be efficiently affinity purified. An antiserum raised against a MISTIC-fusion with the full-length transmembrane nucleoporin POM121 was affinity purified using the MISTIC-POM121 protein coupled to Affigel 10 matrix as bait. The unpurified antiserum (at a 1:1,000 dilution) as well as the affinity purified antibodies (at a 0.1 mg/ml and 0.03 mg/ml dilution) were tested by western blotting after 6% SDS-PAGE of 10 μg, 3 μg and 1 μg of a total membrane fraction from Xenopus egg extracts. Molecular size markers as well as the position of POM121 are indicated.
Figure Legend Snippet: Antisera raised against MISTIC-fusion proteins can be efficiently affinity purified. An antiserum raised against a MISTIC-fusion with the full-length transmembrane nucleoporin POM121 was affinity purified using the MISTIC-POM121 protein coupled to Affigel 10 matrix as bait. The unpurified antiserum (at a 1:1,000 dilution) as well as the affinity purified antibodies (at a 0.1 mg/ml and 0.03 mg/ml dilution) were tested by western blotting after 6% SDS-PAGE of 10 μg, 3 μg and 1 μg of a total membrane fraction from Xenopus egg extracts. Molecular size markers as well as the position of POM121 are indicated.

Techniques Used: Affinity Purification, Western Blot, SDS Page

Related Articles

Centrifugation:

Article Title: Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1
Article Snippet: Pull-down assay The pull-down assay was performed using a flavone-conjugate coupled to Affi-Gel-10 agarose beads (Bio-Rad laboratories, Hercules, CA, USA). .. Cellular debris was removed by centrifugation at 10 000 × g for 30 min. A 500 μ g of total protein extract was incubated for 12 h at 4°C with 40 μ l of flavone-coupled, negative control-coupled or uncoupled Affi-Gel beads.

Injection:

Article Title: ?TrCP-Mediated Proteolysis of NF-?B1 p105 Requires Phosphorylation of p105 Serines 927 and 932
Article Snippet: After high-pressure liquid chromatography purification, this phosphopeptide was coupled to keyhole limpet hemocyanin (Pierce) and injected into rabbits. .. For use in Western blots, immunoglobulin G was isolated from 4 ml of antiserum by using protein G-Sepharose (Amersham Bioscience) and then affinity purified on a column of Affi-Gel 10 (Bio-Rad) coupled to 10 mg of phosphopeptide after first passing through an Affi-Gel 10 column coupled to 10 mg of unphosphorylated peptide from residues 922 to 935.

Expressing:

Article Title: Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover
Article Snippet: .. For co-precipitation of human TDG with SUMO-1 and SUMO-3, crude protein extract from E.coli BL21(DE3) cells expressing either recombinant SUMO-1, SUMO-3 or no human protein was covalently coupled to Affi-Gel®10 beads (Bio-Rad) following the manufacturer’s protocol. ..

Synthesized:

Article Title: ?TrCP-Mediated Proteolysis of NF-?B1 p105 Requires Phosphorylation of p105 Serines 927 and 932
Article Snippet: To generate the anti-phospho-S932 -p105 antibody, a peptide was synthesized corresponding to residues 922 through 935 of human p105 in which serine 932 was phosphorylated. .. For use in Western blots, immunoglobulin G was isolated from 4 ml of antiserum by using protein G-Sepharose (Amersham Bioscience) and then affinity purified on a column of Affi-Gel 10 (Bio-Rad) coupled to 10 mg of phosphopeptide after first passing through an Affi-Gel 10 column coupled to 10 mg of unphosphorylated peptide from residues 922 to 935.

Article Title: Phosphorylation of the immunosuppressant FK506-binding protein FKBP52 by casein kinase II: Regulation of HSP90-binding activity of FKBP52
Article Snippet: FK506 was esterified at the C32-hydroxyl group with an allyloxycarbonyl-protected amino acid, and immobilized onto Affi-Gel 10 (Bio-Rad) after removal of the protective group ( ). .. Pept419 (Phe-135–Gly-149 of rabbit FKBP52) and Pept790 (Lys-182–Pro-201 of rabbit FKBP52) were synthesized and conjugated to keyhole limpet hemocyanin (KLH) ( ).

Construct:

Article Title: ?TrCP-Mediated Proteolysis of NF-?B1 p105 Requires Phosphorylation of p105 Serines 927 and 932
Article Snippet: Paragraph title: cDNA constructs and antibodies. ... For use in Western blots, immunoglobulin G was isolated from 4 ml of antiserum by using protein G-Sepharose (Amersham Bioscience) and then affinity purified on a column of Affi-Gel 10 (Bio-Rad) coupled to 10 mg of phosphopeptide after first passing through an Affi-Gel 10 column coupled to 10 mg of unphosphorylated peptide from residues 922 to 935.

Incubation:

Article Title: Single-stranded DNA catenation mediated by human EVL and a type I topoisomerase
Article Snippet: Pull-down assays with TOPO IIIα-conjugated beads Purified human TOPO IIIα was covalently conjugated to Affi-Gel 10 beads (100 µl, Bio-Rad), according to the manufacturer's instructions. .. To block the remaining active ester sites, ethanolamine (pH 8.0) was added to a final concentration of 100 mM, and the resin was incubated at 4°C overnight.

Article Title: ?TrCP-Mediated Proteolysis of NF-?B1 p105 Requires Phosphorylation of p105 Serines 927 and 932
Article Snippet: .. Therefore, to determine the effectiveness of siRNAs in decreasing βTrCP1 and βTrCP2 levels, siRNAs or control transfected cells were lysed in buffer A (1 ml) and centrifuged lysates were incubated overnight with phospho-S32/S36 ΙκBα peptide (Fig. ) coupled to Affi-Gel 10 beads (Bio-Rad). .. Beads were then washed four times in buffer A and isolated endogenous βTrCP1 and βTrCP2 quantified by Western blotting.

Article Title: Yra1 Is Required for S Phase Entry and Affects Dia2 Binding to Replication Origins ▿
Article Snippet: Supernatant was incubated for 4 h at 4°C with rotation with 1 ml GT-Sepharose (Amersham Pharmacia) equilibrated with lysis buffer. .. The GST-Dia2 protein was coupled to Affigel-10 (Bio-Rad) according to the manufacturer's instructions.

Article Title: Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover
Article Snippet: After incubation for 45 min at 4°C, the beads were washed in 6× 200 µl and 1× 20 µl (last wash) of interaction buffer and then transferred to a new reaction tube. .. For co-precipitation of human TDG with SUMO-1 and SUMO-3, crude protein extract from E.coli BL21(DE3) cells expressing either recombinant SUMO-1, SUMO-3 or no human protein was covalently coupled to Affi-Gel®10 beads (Bio-Rad) following the manufacturer’s protocol.

Article Title: Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities
Article Snippet: The pull-down assay with PSF-conjugated beads PSF (40 µg) was covalently conjugated to Affi-Gel 10 beads (75 µl, Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions, and the Affi-Gel 10-protein matrices were adjusted to 50% slurries. .. The His6 -tagged RAD51, the His6 -tagged RAD51 N-terminal domain, or the His6 -tagged RAD51 ATPase domain (4 µg) was incubated with PSF-conjugated Affi-Gel 10 beads (20 µl, 50% slurry) in 100 µl of binding buffer, containing 20 mM sodium phosphate (pH 8.0), 60 mM NaCl, 0.1 mM EDTA, 2 mM 2-mercaptoethanol, 0.05% Triton X-100 and 10% glycerol.

Article Title: Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1
Article Snippet: Pull-down assay The pull-down assay was performed using a flavone-conjugate coupled to Affi-Gel-10 agarose beads (Bio-Rad laboratories, Hercules, CA, USA). .. Cellular debris was removed by centrifugation at 10 000 × g for 30 min. A 500 μ g of total protein extract was incubated for 12 h at 4°C with 40 μ l of flavone-coupled, negative control-coupled or uncoupled Affi-Gel beads.

Article Title: Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞
Article Snippet: Pulldown Assays with EVL-conjugated Beads —The EVL protein was covalently conjugated to Affi-Gel 10 beads (100 μl; Bio-Rad), according to the manufacturer's instructions. .. To block the remaining active ester sites, ethanolamine (pH 8.0) was added to a final concentration of 100 m m , and the resin was incubated at 4 °C overnight.

Article Title: Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway
Article Snippet: Purified recombinant TRX-CHS or TRX-CHI was covalently attached to Affi-Gel 10 activated resin (Bio-Rad) in 100 mM Mops, pH 7.2/150 mM NaCl/1 mM 2-mercaptoethanol/0.1% Tween-20/80 mM CaCl2 for 4 h at 4°C. .. To prepare the extracts for affinity purification of flavonoid enzymes, seedlings were ground to a fine powder in liquid nitrogen, suspended in plant lysis buffer (50 mM Tris, pH 7.2/150 mM NaCl/1 mM 2-mercaptoethanol/70 μg/ml DNase I/0.6% Nonidet P-40/0.6% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS]/4 mM pefabloc/1 μM aprotinin/1 μM PMSF/4 μM benzamidine/2 μM leupeptin/2 μM pepstatin), and incubated at 25°C for 20 min. Insoluble material was pelleted at 30,000 × g for 10 min at 20°C, and 400 μl of the remaining soluble plant lysate was incubated with 25 nmol of resin-coupled ligand with gentle agitation at 4°C for 2 h. The resin was pelleted at 500 × g for 30 sec and resuspended in 30 vol of plant lysis buffer without DNase.

Mass Spectrometry:

Article Title: Yra1 Is Required for S Phase Entry and Affects Dia2 Binding to Replication Origins ▿
Article Snippet: Paragraph title: Protein purification and mass spectrometry. ... The GST-Dia2 protein was coupled to Affigel-10 (Bio-Rad) according to the manufacturer's instructions.

Modification:

Article Title: Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover
Article Snippet: For co-precipitation of human TDG with SUMO-1 and SUMO-3, crude protein extract from E.coli BL21(DE3) cells expressing either recombinant SUMO-1, SUMO-3 or no human protein was covalently coupled to Affi-Gel®10 beads (Bio-Rad) following the manufacturer’s protocol. .. The beads were then incubated with 600 ng of TDG protein (wild-type, mutant or in vitro modified variants) in 50 µl of interaction buffer for 1 h at 4°C under gentle rotation.

Western Blot:

Article Title: ?TrCP-Mediated Proteolysis of NF-?B1 p105 Requires Phosphorylation of p105 Serines 927 and 932
Article Snippet: .. For use in Western blots, immunoglobulin G was isolated from 4 ml of antiserum by using protein G-Sepharose (Amersham Bioscience) and then affinity purified on a column of Affi-Gel 10 (Bio-Rad) coupled to 10 mg of phosphopeptide after first passing through an Affi-Gel 10 column coupled to 10 mg of unphosphorylated peptide from residues 922 to 935. .. Antibodies recognizing IκBα (sc-371; Santa Cruz), βTrCP1 and βTrCP2 (sc-8863; Santa Cruz), and the Flag epitope (M2 MAb; Sigma) were purchased from the indicated suppliers.

Article Title: ?TrCP-Mediated Proteolysis of NF-?B1 p105 Requires Phosphorylation of p105 Serines 927 and 932
Article Snippet: Endogenous βTrCP1 and βTrCP2 were not detected on the Western blot of total cell lysates. .. Therefore, to determine the effectiveness of siRNAs in decreasing βTrCP1 and βTrCP2 levels, siRNAs or control transfected cells were lysed in buffer A (1 ml) and centrifuged lysates were incubated overnight with phospho-S32/S36 ΙκBα peptide (Fig. ) coupled to Affi-Gel 10 beads (Bio-Rad).

Article Title: Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover
Article Snippet: Bound proteins were eluted by boiling the beads in 20 µl of 2× SDS sample buffer at 95°C for 2 min and analysed further by 15% SDS–PAGE and western blotting. .. For co-precipitation of human TDG with SUMO-1 and SUMO-3, crude protein extract from E.coli BL21(DE3) cells expressing either recombinant SUMO-1, SUMO-3 or no human protein was covalently coupled to Affi-Gel®10 beads (Bio-Rad) following the manufacturer’s protocol.

Chloramphenicol Acetyltransferase Assay:

Article Title: Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞
Article Snippet: For the ssDNA substrate used in the D-loop assay, the following HPLC-purified oligonucleotide was purchased: 50-mer, 5′-GGA ATT CGG TAT TCC CAG GCG GTC TCC CAT CCA AGT ACT AAC CGA GCC CT-3′. .. Pulldown Assays with EVL-conjugated Beads —The EVL protein was covalently conjugated to Affi-Gel 10 beads (100 μl; Bio-Rad), according to the manufacturer's instructions.

Derivative Assay:

Article Title: Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover
Article Snippet: A 9 µg aliquot of E.coli BL21(DE3) extract derived from cells expressing either SUMO-1, SUMO-3 or no human protein was then added to the beads in 200 µl of interaction buffer. .. For co-precipitation of human TDG with SUMO-1 and SUMO-3, crude protein extract from E.coli BL21(DE3) cells expressing either recombinant SUMO-1, SUMO-3 or no human protein was covalently coupled to Affi-Gel®10 beads (Bio-Rad) following the manufacturer’s protocol.

High Performance Liquid Chromatography:

Article Title: ?TrCP-Mediated Proteolysis of NF-?B1 p105 Requires Phosphorylation of p105 Serines 927 and 932
Article Snippet: After high-pressure liquid chromatography purification, this phosphopeptide was coupled to keyhole limpet hemocyanin (Pierce) and injected into rabbits. .. For use in Western blots, immunoglobulin G was isolated from 4 ml of antiserum by using protein G-Sepharose (Amersham Bioscience) and then affinity purified on a column of Affi-Gel 10 (Bio-Rad) coupled to 10 mg of phosphopeptide after first passing through an Affi-Gel 10 column coupled to 10 mg of unphosphorylated peptide from residues 922 to 935.

Article Title: Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞
Article Snippet: For the ssDNA substrate used in the D-loop assay, the following HPLC-purified oligonucleotide was purchased: 50-mer, 5′-GGA ATT CGG TAT TCC CAG GCG GTC TCC CAT CCA AGT ACT AAC CGA GCC CT-3′. .. Pulldown Assays with EVL-conjugated Beads —The EVL protein was covalently conjugated to Affi-Gel 10 beads (100 μl; Bio-Rad), according to the manufacturer's instructions.

Transfection:

Article Title: ?TrCP-Mediated Proteolysis of NF-?B1 p105 Requires Phosphorylation of p105 Serines 927 and 932
Article Snippet: .. Therefore, to determine the effectiveness of siRNAs in decreasing βTrCP1 and βTrCP2 levels, siRNAs or control transfected cells were lysed in buffer A (1 ml) and centrifuged lysates were incubated overnight with phospho-S32/S36 ΙκBα peptide (Fig. ) coupled to Affi-Gel 10 beads (Bio-Rad). .. Beads were then washed four times in buffer A and isolated endogenous βTrCP1 and βTrCP2 quantified by Western blotting.

Concentration Assay:

Article Title: Single-stranded DNA catenation mediated by human EVL and a type I topoisomerase
Article Snippet: Pull-down assays with TOPO IIIα-conjugated beads Purified human TOPO IIIα was covalently conjugated to Affi-Gel 10 beads (100 µl, Bio-Rad), according to the manufacturer's instructions. .. To block the remaining active ester sites, ethanolamine (pH 8.0) was added to a final concentration of 100 mM, and the resin was incubated at 4°C overnight.

Article Title: Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞
Article Snippet: Pulldown Assays with EVL-conjugated Beads —The EVL protein was covalently conjugated to Affi-Gel 10 beads (100 μl; Bio-Rad), according to the manufacturer's instructions. .. To block the remaining active ester sites, ethanolamine (pH 8.0) was added to a final concentration of 100 m m , and the resin was incubated at 4 °C overnight.

Article Title: Capping Protein Binding to S100B: implications for the "tentacle" model for capping the actin filament barbed end *
Article Snippet: .. 3) where [S100B]bound is the amount of rat S100B bound in complex with the bead-coupled ligand; [resin-coupled ligand] is the concentration of either GST- α C28 or GST- β C34 coupled to glutathione-Sepharose, α C28 peptide, or β C34 peptide coupled to Affi-Gel® 10, or CP( α 1 β 1) coupled to Affi-Gel® 15; [S100B] is the rat S100B concentration; and Kd is the equilibrium dissociation constant. .. The amount of S100B bound to the beads was calculated using the equation, [S100B]bound = [S100B]total − [S100B]S/N − [nonspecific binding].

Protease Inhibitor:

Article Title: Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1
Article Snippet: Pull-down assay The pull-down assay was performed using a flavone-conjugate coupled to Affi-Gel-10 agarose beads (Bio-Rad laboratories, Hercules, CA, USA). .. CEM cells (1 × 108 ) were washed in phosphate-buffered saline and lysed in 2 ml lysis buffer containing 50 mM Tris/HCl (pH 8.0), 120 mM NaCl, 1% NP-40, 5 mM DTT, 200 μ M Na-orthovanadate, 25 mM NaF and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany).

Generated:

Article Title: Yra1 Is Required for S Phase Entry and Affects Dia2 Binding to Replication Origins ▿
Article Snippet: The GST-Dia2 protein was coupled to Affigel-10 (Bio-Rad) according to the manufacturer's instructions. .. Spheroplasts were generated by incubating cells in 1 M sorbitol, 50 mM Tris-HCl (pH 7.5), 50 mM MgCl2 , 50 mM CaCl2 , and 1.1 mg Zymolyase 100T per g of cells (wet weight) for 30 min at 30°C.

other:

Article Title: ?TrCP-Mediated Proteolysis of NF-?B1 p105 Requires Phosphorylation of p105 Serines 927 and 932
Article Snippet: To investigate this possibility directly, each of the phosphopeptides was individually coupled to Affi-Gel 10 beads and then used as affinity matrices to purify endogenous βTrCP from HeLa cell lysates.

Article Title: Sonic hedgehog delivery from self-assembled nanofiber hydrogels reduces the fibrotic response in models of erectile dysfunction
Article Snippet: Affi-Gel beads (100–200 mesh) were equilibrated with SHH or BSA (control) protein (0.25μg/μl) overnight at 4°C.

Article Title: Phosphorylation of the immunosuppressant FK506-binding protein FKBP52 by casein kinase II: Regulation of HSP90-binding activity of FKBP52
Article Snippet: The cell lysates were centrifuged at 15,000 × g for 60 min at 2°C and precleared with Affi-Gel 10.

Affinity Purification:

Article Title: ?TrCP-Mediated Proteolysis of NF-?B1 p105 Requires Phosphorylation of p105 Serines 927 and 932
Article Snippet: .. For use in Western blots, immunoglobulin G was isolated from 4 ml of antiserum by using protein G-Sepharose (Amersham Bioscience) and then affinity purified on a column of Affi-Gel 10 (Bio-Rad) coupled to 10 mg of phosphopeptide after first passing through an Affi-Gel 10 column coupled to 10 mg of unphosphorylated peptide from residues 922 to 935. .. Antibodies recognizing IκBα (sc-371; Santa Cruz), βTrCP1 and βTrCP2 (sc-8863; Santa Cruz), and the Flag epitope (M2 MAb; Sigma) were purchased from the indicated suppliers.

Article Title: Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway
Article Snippet: Purified recombinant TRX-CHS or TRX-CHI was covalently attached to Affi-Gel 10 activated resin (Bio-Rad) in 100 mM Mops, pH 7.2/150 mM NaCl/1 mM 2-mercaptoethanol/0.1% Tween-20/80 mM CaCl2 for 4 h at 4°C. .. To prepare the extracts for affinity purification of flavonoid enzymes, seedlings were ground to a fine powder in liquid nitrogen, suspended in plant lysis buffer (50 mM Tris, pH 7.2/150 mM NaCl/1 mM 2-mercaptoethanol/70 μg/ml DNase I/0.6% Nonidet P-40/0.6% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS]/4 mM pefabloc/1 μM aprotinin/1 μM PMSF/4 μM benzamidine/2 μM leupeptin/2 μM pepstatin), and incubated at 25°C for 20 min. Insoluble material was pelleted at 30,000 × g for 10 min at 20°C, and 400 μl of the remaining soluble plant lysate was incubated with 25 nmol of resin-coupled ligand with gentle agitation at 4°C for 2 h. The resin was pelleted at 500 × g for 30 sec and resuspended in 30 vol of plant lysis buffer without DNase.

Recombinant:

Article Title: Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover
Article Snippet: .. For co-precipitation of human TDG with SUMO-1 and SUMO-3, crude protein extract from E.coli BL21(DE3) cells expressing either recombinant SUMO-1, SUMO-3 or no human protein was covalently coupled to Affi-Gel®10 beads (Bio-Rad) following the manufacturer’s protocol. ..

Article Title: Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway
Article Snippet: .. Purified recombinant TRX-CHS or TRX-CHI was covalently attached to Affi-Gel 10 activated resin (Bio-Rad) in 100 mM Mops, pH 7.2/150 mM NaCl/1 mM 2-mercaptoethanol/0.1% Tween-20/80 mM CaCl2 for 4 h at 4°C. ..

Cellular Antioxidant Activity Assay:

Article Title: Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞
Article Snippet: The DNA sequences used in the strand exchange assay with the oligonucleotides are as follows: 63-mer, 5′-TCC TTT TGA TAA GAG GTC ATT TTT GCG GAT GGC TTA GAG CTT AAT TGC TGA ATC TGG TGC TGT-3′; 32-mer top strand, 5′-CCA TCC GCA AAA ATG ACC TCT TAT CAA AAG GA-3′; 32-mer bottom strand, 5′-TCC TTT TGA TAA GAG GTC ATT TTT GCG GAT GG-3′. .. Pulldown Assays with EVL-conjugated Beads —The EVL protein was covalently conjugated to Affi-Gel 10 beads (100 μl; Bio-Rad), according to the manufacturer's instructions.

Nucleic Acid Electrophoresis:

Article Title: Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities
Article Snippet: The pull-down assay with PSF-conjugated beads PSF (40 µg) was covalently conjugated to Affi-Gel 10 beads (75 µl, Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions, and the Affi-Gel 10-protein matrices were adjusted to 50% slurries. .. The proteins bound to the PSF beads were fractionated by 15% SDS-polyacrylamide gel electrophoresis (PAGE), and the bands were visualized by Coomassie Brilliant Blue staining.

Pull Down Assay:

Article Title: Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities
Article Snippet: .. The pull-down assay with PSF-conjugated beads PSF (40 µg) was covalently conjugated to Affi-Gel 10 beads (75 µl, Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions, and the Affi-Gel 10-protein matrices were adjusted to 50% slurries. .. The His6 -tagged RAD51, the His6 -tagged RAD51 N-terminal domain, or the His6 -tagged RAD51 ATPase domain (4 µg) was incubated with PSF-conjugated Affi-Gel 10 beads (20 µl, 50% slurry) in 100 µl of binding buffer, containing 20 mM sodium phosphate (pH 8.0), 60 mM NaCl, 0.1 mM EDTA, 2 mM 2-mercaptoethanol, 0.05% Triton X-100 and 10% glycerol.

Article Title: Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1
Article Snippet: .. Pull-down assay The pull-down assay was performed using a flavone-conjugate coupled to Affi-Gel-10 agarose beads (Bio-Rad laboratories, Hercules, CA, USA). .. CEM cells (1 × 108 ) were washed in phosphate-buffered saline and lysed in 2 ml lysis buffer containing 50 mM Tris/HCl (pH 8.0), 120 mM NaCl, 1% NP-40, 5 mM DTT, 200 μ M Na-orthovanadate, 25 mM NaF and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany).

Mutagenesis:

Article Title: Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover
Article Snippet: For co-precipitation of human TDG with SUMO-1 and SUMO-3, crude protein extract from E.coli BL21(DE3) cells expressing either recombinant SUMO-1, SUMO-3 or no human protein was covalently coupled to Affi-Gel®10 beads (Bio-Rad) following the manufacturer’s protocol. .. The beads were then incubated with 600 ng of TDG protein (wild-type, mutant or in vitro modified variants) in 50 µl of interaction buffer for 1 h at 4°C under gentle rotation.

Isolation:

Article Title: ?TrCP-Mediated Proteolysis of NF-?B1 p105 Requires Phosphorylation of p105 Serines 927 and 932
Article Snippet: .. For use in Western blots, immunoglobulin G was isolated from 4 ml of antiserum by using protein G-Sepharose (Amersham Bioscience) and then affinity purified on a column of Affi-Gel 10 (Bio-Rad) coupled to 10 mg of phosphopeptide after first passing through an Affi-Gel 10 column coupled to 10 mg of unphosphorylated peptide from residues 922 to 935. .. Antibodies recognizing IκBα (sc-371; Santa Cruz), βTrCP1 and βTrCP2 (sc-8863; Santa Cruz), and the Flag epitope (M2 MAb; Sigma) were purchased from the indicated suppliers.

Article Title: ?TrCP-Mediated Proteolysis of NF-?B1 p105 Requires Phosphorylation of p105 Serines 927 and 932
Article Snippet: Therefore, to determine the effectiveness of siRNAs in decreasing βTrCP1 and βTrCP2 levels, siRNAs or control transfected cells were lysed in buffer A (1 ml) and centrifuged lysates were incubated overnight with phospho-S32/S36 ΙκBα peptide (Fig. ) coupled to Affi-Gel 10 beads (Bio-Rad). .. Beads were then washed four times in buffer A and isolated endogenous βTrCP1 and βTrCP2 quantified by Western blotting.

Size-exclusion Chromatography:

Article Title: Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway
Article Snippet: Purified recombinant TRX-CHS or TRX-CHI was covalently attached to Affi-Gel 10 activated resin (Bio-Rad) in 100 mM Mops, pH 7.2/150 mM NaCl/1 mM 2-mercaptoethanol/0.1% Tween-20/80 mM CaCl2 for 4 h at 4°C. .. To prepare the extracts for affinity purification of flavonoid enzymes, seedlings were ground to a fine powder in liquid nitrogen, suspended in plant lysis buffer (50 mM Tris, pH 7.2/150 mM NaCl/1 mM 2-mercaptoethanol/70 μg/ml DNase I/0.6% Nonidet P-40/0.6% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS]/4 mM pefabloc/1 μM aprotinin/1 μM PMSF/4 μM benzamidine/2 μM leupeptin/2 μM pepstatin), and incubated at 25°C for 20 min. Insoluble material was pelleted at 30,000 × g for 10 min at 20°C, and 400 μl of the remaining soluble plant lysate was incubated with 25 nmol of resin-coupled ligand with gentle agitation at 4°C for 2 h. The resin was pelleted at 500 × g for 30 sec and resuspended in 30 vol of plant lysis buffer without DNase.

Labeling:

Article Title: Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞
Article Snippet: The 5′-ends of the oligonucleotide (32-mer bottom strand) were labeled with T4 polynucleotide kinase (New England Biolabs) in the presence of [γ-32 P]ATP at 37 °C for 30 min. All DNA concentrations are expressed in moles of nucleotides. .. Pulldown Assays with EVL-conjugated Beads —The EVL protein was covalently conjugated to Affi-Gel 10 beads (100 μl; Bio-Rad), according to the manufacturer's instructions.

Purification:

Article Title: ?TrCP-Mediated Proteolysis of NF-?B1 p105 Requires Phosphorylation of p105 Serines 927 and 932
Article Snippet: After high-pressure liquid chromatography purification, this phosphopeptide was coupled to keyhole limpet hemocyanin (Pierce) and injected into rabbits. .. For use in Western blots, immunoglobulin G was isolated from 4 ml of antiserum by using protein G-Sepharose (Amersham Bioscience) and then affinity purified on a column of Affi-Gel 10 (Bio-Rad) coupled to 10 mg of phosphopeptide after first passing through an Affi-Gel 10 column coupled to 10 mg of unphosphorylated peptide from residues 922 to 935.

Article Title: Single-stranded DNA catenation mediated by human EVL and a type I topoisomerase
Article Snippet: .. Pull-down assays with TOPO IIIα-conjugated beads Purified human TOPO IIIα was covalently conjugated to Affi-Gel 10 beads (100 µl, Bio-Rad), according to the manufacturer's instructions. .. To block the remaining active ester sites, ethanolamine (pH 8.0) was added to a final concentration of 100 mM, and the resin was incubated at 4°C overnight.

Article Title: Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover
Article Snippet: For co-precipitation of SUMO-1 with human TDG, 1 µg of purified recombinant human TDG or an equimolar amount of recombinant human APE1 was added to 20 µl of interaction buffer-equilibrated magnetic Ni2+ -NTA–agarose beads (Qiagen). .. For co-precipitation of human TDG with SUMO-1 and SUMO-3, crude protein extract from E.coli BL21(DE3) cells expressing either recombinant SUMO-1, SUMO-3 or no human protein was covalently coupled to Affi-Gel®10 beads (Bio-Rad) following the manufacturer’s protocol.

Article Title: Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway
Article Snippet: .. Purified recombinant TRX-CHS or TRX-CHI was covalently attached to Affi-Gel 10 activated resin (Bio-Rad) in 100 mM Mops, pH 7.2/150 mM NaCl/1 mM 2-mercaptoethanol/0.1% Tween-20/80 mM CaCl2 for 4 h at 4°C. ..

Article Title: Phosphorylation of the immunosuppressant FK506-binding protein FKBP52 by casein kinase II: Regulation of HSP90-binding activity of FKBP52
Article Snippet: HSP90 was purified from mouse L5178Y cells ( ) and further purified with a Poros-heparin column (Perseptive Biosystems, Cambridge, MA). .. FK506 was esterified at the C32-hydroxyl group with an allyloxycarbonyl-protected amino acid, and immobilized onto Affi-Gel 10 (Bio-Rad) after removal of the protective group ( ).

Protein Purification:

Article Title: Yra1 Is Required for S Phase Entry and Affects Dia2 Binding to Replication Origins ▿
Article Snippet: Paragraph title: Protein purification and mass spectrometry. ... The GST-Dia2 protein was coupled to Affigel-10 (Bio-Rad) according to the manufacturer's instructions.

Binding Assay:

Article Title: Single-stranded DNA catenation mediated by human EVL and a type I topoisomerase
Article Snippet: Pull-down assays with TOPO IIIα-conjugated beads Purified human TOPO IIIα was covalently conjugated to Affi-Gel 10 beads (100 µl, Bio-Rad), according to the manufacturer's instructions. .. The unbound proteins were removed by washing the Affi-Gel 10-TOPO IIIα beads three times with 500 µl of binding buffer, which contained 20 mM HEPES–NaOH (pH 7.5), 300 mM NaCl, 5 mM 2-mercaptoethanol, 20% glycerol and 0.05% Triton X-100.

Article Title: Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities
Article Snippet: The pull-down assay with PSF-conjugated beads PSF (40 µg) was covalently conjugated to Affi-Gel 10 beads (75 µl, Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions, and the Affi-Gel 10-protein matrices were adjusted to 50% slurries. .. The His6 -tagged RAD51, the His6 -tagged RAD51 N-terminal domain, or the His6 -tagged RAD51 ATPase domain (4 µg) was incubated with PSF-conjugated Affi-Gel 10 beads (20 µl, 50% slurry) in 100 µl of binding buffer, containing 20 mM sodium phosphate (pH 8.0), 60 mM NaCl, 0.1 mM EDTA, 2 mM 2-mercaptoethanol, 0.05% Triton X-100 and 10% glycerol.

Article Title: Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞
Article Snippet: Pulldown Assays with EVL-conjugated Beads —The EVL protein was covalently conjugated to Affi-Gel 10 beads (100 μl; Bio-Rad), according to the manufacturer's instructions. .. The unbound proteins were removed by washing the Affi-Gel 10-EVL beads three times with 500 μl of binding buffer, which contained 20 m m HEPES-NaOH (pH 7.3), 100 m m NaCl, 5 m m 2-mercaptoethanol, 30% glycerol, and 0.05% Triton X-100.

Article Title: Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway
Article Snippet: Purified recombinant TRX-CHS or TRX-CHI was covalently attached to Affi-Gel 10 activated resin (Bio-Rad) in 100 mM Mops, pH 7.2/150 mM NaCl/1 mM 2-mercaptoethanol/0.1% Tween-20/80 mM CaCl2 for 4 h at 4°C. .. Coupling efficiency was assessed based on the amount of protein present in solution before and after binding as determined using the Bio-Rad protein assay.

Immunoprecipitation:

Article Title: ?TrCP-Mediated Proteolysis of NF-?B1 p105 Requires Phosphorylation of p105 Serines 927 and 932
Article Snippet: Endogenous p105 was immunoprecipitated and detected in Western blots by using the anti-p105C antibody, which recognizes the C terminus of human p105 ( ). .. For use in Western blots, immunoglobulin G was isolated from 4 ml of antiserum by using protein G-Sepharose (Amersham Bioscience) and then affinity purified on a column of Affi-Gel 10 (Bio-Rad) coupled to 10 mg of phosphopeptide after first passing through an Affi-Gel 10 column coupled to 10 mg of unphosphorylated peptide from residues 922 to 935.

Blocking Assay:

Article Title: Single-stranded DNA catenation mediated by human EVL and a type I topoisomerase
Article Snippet: Pull-down assays with TOPO IIIα-conjugated beads Purified human TOPO IIIα was covalently conjugated to Affi-Gel 10 beads (100 µl, Bio-Rad), according to the manufacturer's instructions. .. To block the remaining active ester sites, ethanolamine (pH 8.0) was added to a final concentration of 100 mM, and the resin was incubated at 4°C overnight.

Article Title: Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞
Article Snippet: Pulldown Assays with EVL-conjugated Beads —The EVL protein was covalently conjugated to Affi-Gel 10 beads (100 μl; Bio-Rad), according to the manufacturer's instructions. .. To block the remaining active ester sites, ethanolamine (pH 8.0) was added to a final concentration of 100 m m , and the resin was incubated at 4 °C overnight.

Polyacrylamide Gel Electrophoresis:

Article Title: Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities
Article Snippet: The pull-down assay with PSF-conjugated beads PSF (40 µg) was covalently conjugated to Affi-Gel 10 beads (75 µl, Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions, and the Affi-Gel 10-protein matrices were adjusted to 50% slurries. .. The proteins bound to the PSF beads were fractionated by 15% SDS-polyacrylamide gel electrophoresis (PAGE), and the bands were visualized by Coomassie Brilliant Blue staining.

Lysis:

Article Title: Yra1 Is Required for S Phase Entry and Affects Dia2 Binding to Replication Origins ▿
Article Snippet: GST-Dia2 was eluted in three successive incubations with 1 volume each of lysis buffer containing 15 mM glutathione. .. The GST-Dia2 protein was coupled to Affigel-10 (Bio-Rad) according to the manufacturer's instructions.

Article Title: Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1
Article Snippet: Pull-down assay The pull-down assay was performed using a flavone-conjugate coupled to Affi-Gel-10 agarose beads (Bio-Rad laboratories, Hercules, CA, USA). .. CEM cells (1 × 108 ) were washed in phosphate-buffered saline and lysed in 2 ml lysis buffer containing 50 mM Tris/HCl (pH 8.0), 120 mM NaCl, 1% NP-40, 5 mM DTT, 200 μ M Na-orthovanadate, 25 mM NaF and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany).

Article Title: Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway
Article Snippet: Purified recombinant TRX-CHS or TRX-CHI was covalently attached to Affi-Gel 10 activated resin (Bio-Rad) in 100 mM Mops, pH 7.2/150 mM NaCl/1 mM 2-mercaptoethanol/0.1% Tween-20/80 mM CaCl2 for 4 h at 4°C. .. To prepare the extracts for affinity purification of flavonoid enzymes, seedlings were ground to a fine powder in liquid nitrogen, suspended in plant lysis buffer (50 mM Tris, pH 7.2/150 mM NaCl/1 mM 2-mercaptoethanol/70 μg/ml DNase I/0.6% Nonidet P-40/0.6% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS]/4 mM pefabloc/1 μM aprotinin/1 μM PMSF/4 μM benzamidine/2 μM leupeptin/2 μM pepstatin), and incubated at 25°C for 20 min. Insoluble material was pelleted at 30,000 × g for 10 min at 20°C, and 400 μl of the remaining soluble plant lysate was incubated with 25 nmol of resin-coupled ligand with gentle agitation at 4°C for 2 h. The resin was pelleted at 500 × g for 30 sec and resuspended in 30 vol of plant lysis buffer without DNase.

Activated Clotting Time Assay:

Article Title: Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞
Article Snippet: For the ssDNA substrate used in the D-loop assay, the following HPLC-purified oligonucleotide was purchased: 50-mer, 5′-GGA ATT CGG TAT TCC CAG GCG GTC TCC CAT CCA AGT ACT AAC CGA GCC CT-3′. .. Pulldown Assays with EVL-conjugated Beads —The EVL protein was covalently conjugated to Affi-Gel 10 beads (100 μl; Bio-Rad), according to the manufacturer's instructions.

SDS Page:

Article Title: Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover
Article Snippet: Bound proteins were eluted by boiling the beads in 20 µl of 2× SDS sample buffer at 95°C for 2 min and analysed further by 15% SDS–PAGE and western blotting. .. For co-precipitation of human TDG with SUMO-1 and SUMO-3, crude protein extract from E.coli BL21(DE3) cells expressing either recombinant SUMO-1, SUMO-3 or no human protein was covalently coupled to Affi-Gel®10 beads (Bio-Rad) following the manufacturer’s protocol.

Plasmid Preparation:

Article Title: Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞
Article Snippet: The pB5Sarray DNA contained 11 repeats of a sea urchin 5 S rRNA gene (207-bp fragment) within the pBlueScript II SK(+) vector. .. Pulldown Assays with EVL-conjugated Beads —The EVL protein was covalently conjugated to Affi-Gel 10 beads (100 μl; Bio-Rad), according to the manufacturer's instructions.

Affinity Chromatography:

Article Title: Redistribution of Cyclophilin A to Viral Factories during Vaccinia Virus Infection and Its Incorporation into Mature Particles
Article Snippet: Paragraph title: Affinity chromatography. ... Anhydrous coupling of 8′A-Cs to Affi-Gel 10 (Bio-Rad) resin was carried out essentially as previously described ( ).

Article Title: Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway
Article Snippet: Paragraph title: Affinity Chromatography. ... Purified recombinant TRX-CHS or TRX-CHI was covalently attached to Affi-Gel 10 activated resin (Bio-Rad) in 100 mM Mops, pH 7.2/150 mM NaCl/1 mM 2-mercaptoethanol/0.1% Tween-20/80 mM CaCl2 for 4 h at 4°C.

In Vitro:

Article Title: Modification of the human thymine-DNA glycosylase by ubiquitin-like proteins facilitates enzymatic turnover
Article Snippet: For co-precipitation of human TDG with SUMO-1 and SUMO-3, crude protein extract from E.coli BL21(DE3) cells expressing either recombinant SUMO-1, SUMO-3 or no human protein was covalently coupled to Affi-Gel®10 beads (Bio-Rad) following the manufacturer’s protocol. .. The beads were then incubated with 600 ng of TDG protein (wild-type, mutant or in vitro modified variants) in 50 µl of interaction buffer for 1 h at 4°C under gentle rotation.

FLAG-tag:

Article Title: ?TrCP-Mediated Proteolysis of NF-?B1 p105 Requires Phosphorylation of p105 Serines 927 and 932
Article Snippet: For use in Western blots, immunoglobulin G was isolated from 4 ml of antiserum by using protein G-Sepharose (Amersham Bioscience) and then affinity purified on a column of Affi-Gel 10 (Bio-Rad) coupled to 10 mg of phosphopeptide after first passing through an Affi-Gel 10 column coupled to 10 mg of unphosphorylated peptide from residues 922 to 935. .. Antibodies recognizing IκBα (sc-371; Santa Cruz), βTrCP1 and βTrCP2 (sc-8863; Santa Cruz), and the Flag epitope (M2 MAb; Sigma) were purchased from the indicated suppliers.

CTG Assay:

Article Title: Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞
Article Snippet: For the ssDNA annealing assay, the following high performance liquid chromatography (HPLC)-purified oligonucleotides were purchased from Nihon Gene Research Laboratory: 5′-GTC CCA GGC CAT TAC AGA TCA ATC CTG AGC ATG TTT ACC AAG CGC ATT G-3′ and 5′-CAA TGC GCT TGG TAA ACA TGC TCA GGA TTG ATC TGT AAT GGC CTG GGA C-3′. .. Pulldown Assays with EVL-conjugated Beads —The EVL protein was covalently conjugated to Affi-Gel 10 beads (100 μl; Bio-Rad), according to the manufacturer's instructions.

Staining:

Article Title: Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities
Article Snippet: The pull-down assay with PSF-conjugated beads PSF (40 µg) was covalently conjugated to Affi-Gel 10 beads (75 µl, Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions, and the Affi-Gel 10-protein matrices were adjusted to 50% slurries. .. The proteins bound to the PSF beads were fractionated by 15% SDS-polyacrylamide gel electrophoresis (PAGE), and the bands were visualized by Coomassie Brilliant Blue staining.

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  • 94
    Bio-Rad sepharose beads
    The LU of AtRMR1 binds to the CTPP peptide of phaseolin. (A) Peptide sequences. (B) In vitro binding assay. The in vitro binding assay was performed as described in Materials and methods. Peptides immobilized on <t>Sepharose</t> beads were incubated with purified GST-LU or GST alone under different pH conditions at 4°C. Sepharose beads were washed three times with binding buffer, and bound proteins were eluted three times with elution buffer. The unbound (Un), wash (Wa), and eluted (El) fractions were analyzed by Western blot analysis using anti-GST antibody. In, input GST-LU.
    Sepharose Beads, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Bio-Rad affigel 10 slurry resin
    Src kinase signaling is mediated by TLR4–Src interaction. Membranous protein fractions were immunoprecipitated by <t>Affigel</t> 10 resin–TLR4 antibody conjugate and analyzed by Western blot. a p-TLR4 level expressed as a ratio of p-TLR4 to that
    Affigel 10 Slurry Resin, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad affigel 10 beads
    The last six C-terminal amino acids and the N-myristoyl moiety are required for the binding of p22 to GAPDH ( A ) Upper panel: <t>Affigel-10</t> beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of myr-p22-Δ190-195 (lanes 4–6). Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or myr-p22-Δ190-195 (lanes 3). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 or myr-p22-Δ190-195 (0.01 μg each) was used as the control. ( B ) Upper panel: Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or p22-rec (lanes 3). Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of p22-rec (lanes 4–6). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 (0.05 μg) or p22-rec (0.01 μg) was used as the control. ( A , B ) Lower panels: quantification of relative percentage of myr-p22, myr-p22-Δ190-195 or p22-rec binding to GAPDH- or ovalbumin-beads. In ( A , B ) lanes 1–6 in upper panels correspond to lanes 1–6 in lower panels. In ( A , B ) lower panels, the amount of myr-p22 associated with GAPDH-coupled beads when 4 μg of myr-p22 was incubated with 50 μg of GAPDH is considered as 100%.
    Affigel 10 Beads, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The LU of AtRMR1 binds to the CTPP peptide of phaseolin. (A) Peptide sequences. (B) In vitro binding assay. The in vitro binding assay was performed as described in Materials and methods. Peptides immobilized on Sepharose beads were incubated with purified GST-LU or GST alone under different pH conditions at 4°C. Sepharose beads were washed three times with binding buffer, and bound proteins were eluted three times with elution buffer. The unbound (Un), wash (Wa), and eluted (El) fractions were analyzed by Western blot analysis using anti-GST antibody. In, input GST-LU.

    Journal: The Journal of Cell Biology

    Article Title: AtRMR1 functions as a cargo receptor for protein trafficking to the protein storage vacuole

    doi: 10.1083/jcb.200504112

    Figure Lengend Snippet: The LU of AtRMR1 binds to the CTPP peptide of phaseolin. (A) Peptide sequences. (B) In vitro binding assay. The in vitro binding assay was performed as described in Materials and methods. Peptides immobilized on Sepharose beads were incubated with purified GST-LU or GST alone under different pH conditions at 4°C. Sepharose beads were washed three times with binding buffer, and bound proteins were eluted three times with elution buffer. The unbound (Un), wash (Wa), and eluted (El) fractions were analyzed by Western blot analysis using anti-GST antibody. In, input GST-LU.

    Article Snippet: In vitro binding assay of the LU of AtRMR1 with peptides Five peptides ( A) were chemically synthesized (Anygen Inc.) and immobilized on Sepharose beads (AffiGel 10) in 10 mM citrate, pH 11.0, for pCTPP, pCTPP(rev), and pCTPPΔ(AFVY) and 10 mM MES, pH 6.0, for pNTPP and pNTPP(rev) according to the manufacturer's instructions (Bio-Rad Laboratories).

    Techniques: In Vitro, Binding Assay, Incubation, Purification, Western Blot

    Src kinase signaling is mediated by TLR4–Src interaction. Membranous protein fractions were immunoprecipitated by Affigel 10 resin–TLR4 antibody conjugate and analyzed by Western blot. a p-TLR4 level expressed as a ratio of p-TLR4 to that

    Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

    Article Title: Alcohol-Induced Interactive Phosphorylation of Src and Toll-like Receptor Regulates the Secretion of Inflammatory Mediators by Human Astrocytes

    doi: 10.1007/s11481-010-9213-z

    Figure Lengend Snippet: Src kinase signaling is mediated by TLR4–Src interaction. Membranous protein fractions were immunoprecipitated by Affigel 10 resin–TLR4 antibody conjugate and analyzed by Western blot. a p-TLR4 level expressed as a ratio of p-TLR4 to that

    Article Snippet: Briefly, Affigel 10 slurry resin (Bio-Rad, Hercules, CA, USA) was activated by three washes with cold de-ionized water.

    Techniques: Immunoprecipitation, Western Blot

    Activation of cPLA2 is mediated by Src kinase signaling. Cytosolic protein fractions were immunoprecipitated by Affigel 10 resin–cPLA2 antibody conjugate and analyzed by Western blot for the expression of p-cPLA2, cPLA2, and p-Src protein. a phosphorylated

    Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

    Article Title: Alcohol-Induced Interactive Phosphorylation of Src and Toll-like Receptor Regulates the Secretion of Inflammatory Mediators by Human Astrocytes

    doi: 10.1007/s11481-010-9213-z

    Figure Lengend Snippet: Activation of cPLA2 is mediated by Src kinase signaling. Cytosolic protein fractions were immunoprecipitated by Affigel 10 resin–cPLA2 antibody conjugate and analyzed by Western blot for the expression of p-cPLA2, cPLA2, and p-Src protein. a phosphorylated

    Article Snippet: Briefly, Affigel 10 slurry resin (Bio-Rad, Hercules, CA, USA) was activated by three washes with cold de-ionized water.

    Techniques: Activation Assay, Immunoprecipitation, Western Blot, Expressing

    The last six C-terminal amino acids and the N-myristoyl moiety are required for the binding of p22 to GAPDH ( A ) Upper panel: Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of myr-p22-Δ190-195 (lanes 4–6). Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or myr-p22-Δ190-195 (lanes 3). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 or myr-p22-Δ190-195 (0.01 μg each) was used as the control. ( B ) Upper panel: Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or p22-rec (lanes 3). Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of p22-rec (lanes 4–6). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 (0.05 μg) or p22-rec (0.01 μg) was used as the control. ( A , B ) Lower panels: quantification of relative percentage of myr-p22, myr-p22-Δ190-195 or p22-rec binding to GAPDH- or ovalbumin-beads. In ( A , B ) lanes 1–6 in upper panels correspond to lanes 1–6 in lower panels. In ( A , B ) lower panels, the amount of myr-p22 associated with GAPDH-coupled beads when 4 μg of myr-p22 was incubated with 50 μg of GAPDH is considered as 100%.

    Journal: Biochemical Journal

    Article Title: Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules

    doi: 10.1042/BJ20040622

    Figure Lengend Snippet: The last six C-terminal amino acids and the N-myristoyl moiety are required for the binding of p22 to GAPDH ( A ) Upper panel: Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of myr-p22-Δ190-195 (lanes 4–6). Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or myr-p22-Δ190-195 (lanes 3). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 or myr-p22-Δ190-195 (0.01 μg each) was used as the control. ( B ) Upper panel: Affigel-10 beads coupled with ovalbumin (lanes 1 and 3) were incubated in the presence of 4 μg of myr-p22 (lanes 1) or p22-rec (lanes 3). Affigel-10 beads coupled with GAPDH (lanes 2, 4–6) were incubated in the presence of 4 μg of myr-p22 (lanes 2) or 2, 4 or 8 μg of p22-rec (lanes 4–6). Equal amounts of beads were assayed by immunoblotting with anti-p22. myr-p22 (0.05 μg) or p22-rec (0.01 μg) was used as the control. ( A , B ) Lower panels: quantification of relative percentage of myr-p22, myr-p22-Δ190-195 or p22-rec binding to GAPDH- or ovalbumin-beads. In ( A , B ) lanes 1–6 in upper panels correspond to lanes 1–6 in lower panels. In ( A , B ) lower panels, the amount of myr-p22 associated with GAPDH-coupled beads when 4 μg of myr-p22 was incubated with 50 μg of GAPDH is considered as 100%.

    Article Snippet: We have analysed the effect of deleting the N-myristoyl group or the last six C-terminal amino acid residues of p22 on its ability to bind GAPDH. myr-p22-Δ190-195 ( A) or p22-rec ( B) did not bind GAPDH coupled with Affigel-10 beads, even when double the amount of these mutants are used, compared with that of the myr-p22 (upper panels, lane 6 versus lane 2).

    Techniques: Binding Assay, Incubation

    p22 interacts directly with GAPDH in vitro ( A ) Blot overlays were performed on 5 μg of purified rabbit muscle GAPDH, BSA or ovalbumin (Ovalb.) in the presence (+) or absence (–) of myr-p22. ( B ) Affigel-10 beads coupled with GAPDH, BSA or aldolase were incubated in the presence (+) or absence (–) of myr-p22. The amount of myr-p22 bound to the beads was detected by immunoblotting using anti-p22. ( C ) K D analysis of the binding of myr-p22 to GAPDH was performed by incubating the indicated concentrations of myr-p22 (molar amounts) with 50 μg of GAPDH-coupled with beads. The amount of bound myr-p22 was detected by immunoblotting with anti-p22. The values for K D and B max were obtained from three repeat experiments as described in the Experimental section.

    Journal: Biochemical Journal

    Article Title: Interactions among p22, glyceraldehyde-3-phosphate dehydrogenase and microtubules

    doi: 10.1042/BJ20040622

    Figure Lengend Snippet: p22 interacts directly with GAPDH in vitro ( A ) Blot overlays were performed on 5 μg of purified rabbit muscle GAPDH, BSA or ovalbumin (Ovalb.) in the presence (+) or absence (–) of myr-p22. ( B ) Affigel-10 beads coupled with GAPDH, BSA or aldolase were incubated in the presence (+) or absence (–) of myr-p22. The amount of myr-p22 bound to the beads was detected by immunoblotting using anti-p22. ( C ) K D analysis of the binding of myr-p22 to GAPDH was performed by incubating the indicated concentrations of myr-p22 (molar amounts) with 50 μg of GAPDH-coupled with beads. The amount of bound myr-p22 was detected by immunoblotting with anti-p22. The values for K D and B max were obtained from three repeat experiments as described in the Experimental section.

    Article Snippet: We have analysed the effect of deleting the N-myristoyl group or the last six C-terminal amino acid residues of p22 on its ability to bind GAPDH. myr-p22-Δ190-195 ( A) or p22-rec ( B) did not bind GAPDH coupled with Affigel-10 beads, even when double the amount of these mutants are used, compared with that of the myr-p22 (upper panels, lane 6 versus lane 2).

    Techniques: In Vitro, Purification, Incubation, Binding Assay