Structured Review

Bio-Rad affigel 10
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 <t>Affigel-10.</t> 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.
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Images

1) 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

2) 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:

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

3) 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

Related Articles

Clone Assay:

Article Title: Cdk1 phosphorylation of Esp1/Separase functions with PP2A and Slk19 to regulate pericentric Cohesin and anaphase onset
Article Snippet: Rabbit serum was harvested, and the α-Mcd1 antibodies purified on an Affigel-10 (Bio-rad) column coupled to purified malE-Mcd1201–301 . malE-Mcd1201–301 was expressed from the plasmid pAR1117 which contains Mcd1201–301 cloned as a BamH1/Sal1 fragment into pMAL-c2 (NEB). α-Slk19 antibodies were generated as follow: coding sequence for the truncated protein Slk19700–817 was amplified by PCR and cloned into pGEX6P-1 (Promega) as a BamHI/EcoRI fragment to create pAR1230. .. Rabbit serum was harvested, α-GST antibodies were removed on an Affigel-10 (Bio-rad) column coupled to GST, and the α-Slk19 antibodies were purified on an Affigel-10 (Bio-rad) column coupled to purified GST-Slk19700–817 .

Article Title: A Wee1 checkpoint inhibits anaphase onset
Article Snippet: Rabbit serum was harvested, clarified by centrifugation, and loaded on Affigel-10 (Bio-Rad Laboratories) columns coupled to purified malE-Clb5, malE-Pds1, or malE-Sgo1, respectively. malE-fusion proteins were expressed from the plasmids pAR651, pAR652, and pAR724, which contain the same fragments listed above cloned as BamH1–Sal1 fragments into pMAL-c2 (New England Biolabs, Inc.). .. Plasmids GST-Swe12-312 (in pGEX-4T3, pAR622), malE-Swe12-819 (in pMAL-c2, pAR623), and His6 -Cdk12-298 (pAR727; gifts of Vu Thai and Doug Kellogg, UCSC, Santa Cruz, Santa Cruz, CA; ) were used to produce GST-Swe1 and His6 -Cdk1 protein for injection into rabbits as described above. α-Swe1 antibodies were purified on Affigel-10 (Bio-Rad Laboratories) columns coupled to purified malE-Swe1. α-Cdk1 antibodies were purified using an Affigel-15 (Bio-Rad Laboratories) column coupled to purified His6 -Cdk1, which is insoluble, so the coupling was done in the presence of 0.3% SDS. α-GFP serum (a gift of Aaron Straight [Stanford University, Stanford, CA] and Andrew Murray [Harvard University, Cambridge, MA]) was generated by immunizing rabbits with bacterially expressed His6 -GFP (pAFS97), and purified as above using an Affigel-10 (Bio-Rad Laboratories) column coupled to purified His6 -GFP. α-Cdc20 antibody was generated as described previously ( ).

Article Title: Somatic copy number changes in DPYD are associated with lower risk of recurrence in triple-negative breast cancers
Article Snippet: Human DPYD cDNA was cloned into the Nco I/Bgl II restriction sites of a pQE-60 vector (Qiagen) including a polyhistidine (His6 )-tag at the C-terminus. .. Anti-DPD antibodies were affinity-purified by coupling the immunogen preparation to a mixture of 50%/50% AffiGel-10 and AffiGel-15 (BioRad, München, Germany).

Centrifugation:

Article Title: A Wee1 checkpoint inhibits anaphase onset
Article Snippet: Rabbit serum was harvested, clarified by centrifugation, and loaded on Affigel-10 (Bio-Rad Laboratories) columns coupled to purified malE-Clb5, malE-Pds1, or malE-Sgo1, respectively. malE-fusion proteins were expressed from the plasmids pAR651, pAR652, and pAR724, which contain the same fragments listed above cloned as BamH1–Sal1 fragments into pMAL-c2 (New England Biolabs, Inc.). .. Plasmids GST-Swe12-312 (in pGEX-4T3, pAR622), malE-Swe12-819 (in pMAL-c2, pAR623), and His6 -Cdk12-298 (pAR727; gifts of Vu Thai and Doug Kellogg, UCSC, Santa Cruz, Santa Cruz, CA; ) were used to produce GST-Swe1 and His6 -Cdk1 protein for injection into rabbits as described above. α-Swe1 antibodies were purified on Affigel-10 (Bio-Rad Laboratories) columns coupled to purified malE-Swe1. α-Cdk1 antibodies were purified using an Affigel-15 (Bio-Rad Laboratories) column coupled to purified His6 -Cdk1, which is insoluble, so the coupling was done in the presence of 0.3% SDS. α-GFP serum (a gift of Aaron Straight [Stanford University, Stanford, CA] and Andrew Murray [Harvard University, Cambridge, MA]) was generated by immunizing rabbits with bacterially expressed His6 -GFP (pAFS97), and purified as above using an Affigel-10 (Bio-Rad Laboratories) column coupled to purified His6 -GFP. α-Cdc20 antibody was generated as described previously ( ).

Article Title: Acetylation of histone H4 lysine 5 and 12 is required for CENP-A deposition into centromeres
Article Snippet: The cell lysate was clarified by centrifugation at 100,000g for 1 h and purified on glutathione agarose (Sigma). .. Purified GST fusions were then coupled to Affigel-10 (Bio-Rad) at a ratio of 3.5 mg protein per ml of resin to generate affinity columns.

Article Title: Non-glycanated Decorin Is a Drug Target on Human Adipose Stromal Cells
Article Snippet: Cells were disrupted in PBS containing protease inhibitors (PI) cocktail with a Dounce homogenizer; centrifugation (15,000 × g for 30 min at 4°C) was performed to separate soluble proteins from membrane pellet. .. Six milligrams of a cysteine-cyclized peptide was coupled onto 250 μL of Affigel 10 (Bio-Rad), and the column was equilibrated with column buffer containing 1% Triton X-100.

Amplification:

Article Title: Cdk1 phosphorylation of Esp1/Separase functions with PP2A and Slk19 to regulate pericentric Cohesin and anaphase onset
Article Snippet: Rabbit serum was harvested, and the α-Mcd1 antibodies purified on an Affigel-10 (Bio-rad) column coupled to purified malE-Mcd1201–301 . malE-Mcd1201–301 was expressed from the plasmid pAR1117 which contains Mcd1201–301 cloned as a BamH1/Sal1 fragment into pMAL-c2 (NEB). α-Slk19 antibodies were generated as follow: coding sequence for the truncated protein Slk19700–817 was amplified by PCR and cloned into pGEX6P-1 (Promega) as a BamHI/EcoRI fragment to create pAR1230. .. Rabbit serum was harvested, α-GST antibodies were removed on an Affigel-10 (Bio-rad) column coupled to GST, and the α-Slk19 antibodies were purified on an Affigel-10 (Bio-rad) column coupled to purified GST-Slk19700–817 .

Article Title: A Wee1 checkpoint inhibits anaphase onset
Article Snippet: Coding sequences for the truncated proteins Clb52-137 , Pds1178-373 , and Sgo1129-326 were amplified using PCR and cloned into pGEX6P-1 (Promega) as BamH1–EcoR1 fragments to create pAR627, pAR624, and pAR717, respectively. .. Plasmids GST-Swe12-312 (in pGEX-4T3, pAR622), malE-Swe12-819 (in pMAL-c2, pAR623), and His6 -Cdk12-298 (pAR727; gifts of Vu Thai and Doug Kellogg, UCSC, Santa Cruz, Santa Cruz, CA; ) were used to produce GST-Swe1 and His6 -Cdk1 protein for injection into rabbits as described above. α-Swe1 antibodies were purified on Affigel-10 (Bio-Rad Laboratories) columns coupled to purified malE-Swe1. α-Cdk1 antibodies were purified using an Affigel-15 (Bio-Rad Laboratories) column coupled to purified His6 -Cdk1, which is insoluble, so the coupling was done in the presence of 0.3% SDS. α-GFP serum (a gift of Aaron Straight [Stanford University, Stanford, CA] and Andrew Murray [Harvard University, Cambridge, MA]) was generated by immunizing rabbits with bacterially expressed His6 -GFP (pAFS97), and purified as above using an Affigel-10 (Bio-Rad Laboratories) column coupled to purified His6 -GFP. α-Cdc20 antibody was generated as described previously ( ).

Expressing:

Article Title: Acetylation of histone H4 lysine 5 and 12 is required for CENP-A deposition into centromeres
Article Snippet: Paragraph title: Protein expression and purification ... Purified GST fusions were then coupled to Affigel-10 (Bio-Rad) at a ratio of 3.5 mg protein per ml of resin to generate affinity columns.

Article Title: Somatic copy number changes in DPYD are associated with lower risk of recurrence in triple-negative breast cancers
Article Snippet: Anti-DPD antibodies were affinity-purified by coupling the immunogen preparation to a mixture of 50%/50% AffiGel-10 and AffiGel-15 (BioRad, München, Germany). .. All batches were checked by one-site ELISA using DPD coated on microplates.

Affinity Purification:

Article Title: An Sp1 transcription factor coordinates caspase-dependent and -independent apoptotic pathways
Article Snippet: Antisera were generated by Pocono Rabbit Farm and Laboratory. .. Polyclonal antibodies were affinity-purified using identical SPTF-3 fragments fused to maltose-binding protein (MBP) and coupled to Affigel 10 (Bio-Rad). .. Chromatin immunoprecipitations were performed as described .

Article Title: Segregation of molecules at cell division reveals native protein localization
Article Snippet: Antibodies to ClpX were purified using Affigel-10 (Bio-Rad) resin. .. The E. coli ClpX protein was purified according to previous protocols , conjugated to resin, and ClpX polyclonal rabbit antibodies (Covance) were purified according to the manufacture's protocol.

Article Title: Somatic copy number changes in DPYD are associated with lower risk of recurrence in triple-negative breast cancers
Article Snippet: Following dialysis and re-naturation in phosphate-buffered saline, 1 mM DTT, pH 7.4, two rabbits were immunised with this protein preparation. .. Anti-DPD antibodies were affinity-purified by coupling the immunogen preparation to a mixture of 50%/50% AffiGel-10 and AffiGel-15 (BioRad, München, Germany). .. Elution was performed with 0.1 M glycine/HCl buffer pH 2.4, followed by re-neutralisation to pH 7.4.

Autoradiography:

Article Title: Cdk1 phosphorylation of Esp1/Separase functions with PP2A and Slk19 to regulate pericentric Cohesin and anaphase onset
Article Snippet: Rabbit serum was harvested, α-GST antibodies were removed on an Affigel-10 (Bio-rad) column coupled to GST, and the α-Slk19 antibodies were purified on an Affigel-10 (Bio-rad) column coupled to purified GST-Slk19700–817 . .. HRP-conjugated α-rabbit and α-mouse secondary antibodies (Bio-rad) were used at a 1:5000 dilution in TBS-T + 4% Fat Free Milk Powder for 30 min to 1 hr., washed with TBS-T and incubated in Western Lightning Plus-ECL (PerkinElmer).

Blocking Assay:

Article Title: Non-glycanated Decorin Is a Drug Target on Human Adipose Stromal Cells
Article Snippet: Six milligrams of a cysteine-cyclized peptide was coupled onto 250 μL of Affigel 10 (Bio-Rad), and the column was equilibrated with column buffer containing 1% Triton X-100. .. Elution was performed with SDS-PAGE loading buffer.

Article Title: Phosphoserine aminotransferase 1 is associated to poor outcome on tamoxifen therapy in recurrent breast cancer
Article Snippet: PSAT1-linked column was prepared by mixing ~2 mL of AffiGel-10® and AffiGel-15 (BioRad) in a 1:1 ratio. .. A total of 0.5 mg of soluble recombinant PSAT1 dissolved in 5 mL of 50 mM HEPES was mixed with the sorbent and incubated overnight at 4 °C.

Article Title: Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA
Article Snippet: Purified His-Pol IV was dialyzed against 100 mM HEPES–NaOH pH 7.4 and 200 mM NaCl at 4°C. .. The coupling reaction was performed by addition of 4.7 mg of His-Pol IV to 0.5 ml AffiGel 10 (Bio-Rad) and mixing for 4 h, followed by a blocking reaction in 20 mM ethanolamine-HCl for 1 h. Pol IV-crosslinked beads were packed into a column (0.5 ml) and were equilibrated in Buffer A (50 mM HEPES–NaOH pH 7.4, 100 mM KCl, 5% glycerol and 1 mM dithiothreitol) as described ( ). .. Escherichia coli ΔdinB strain MK7003 (MG1655 rpsL (Smr ) ΔdinB , laboratory stock) cells were grown at 37°C in LB medium until the OD600 reached ∼0.8 and were then harvested, and 3 g of the cells were lysed by sonication in 90 ml Buffer A supplemented with 15 mM MgCl2 in the presence of 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mg/ml lysozyme and 2.3 U/ml benzonase (Novagen).

Suction Filtration:

Article Title: Identification of Enolase as the Target of 2-Aminothiazoles in Mycobacterium tuberculosis
Article Snippet: AffiGel 10 (Bio-Rad, 0.01 mmol/mL loading) (2 mL, 0.02 mmol) was washed with five portions of cold isopropanol then added to a solution of 4-((2-azethoxy)triethyleneglycol)-N -(4-pyridin-2-yl)thiazol-2-yl)benzamide (33 mg, 70 μmol) and triethylamine (0.1 mL, 0.73 mmol) in dimethyl sulfoxide (2 mL). .. The resulting suspension was vortexed for 24 h, at which point LCMS indicated that the free amine had been consumed.

Article Title: Identification of Enolase as the Target of 2-Aminothiazoles in Mycobacterium tuberculosis
Article Snippet: To generate a control resin, AffiGel 10 (Bio-Rad, 0.01 mmol/mL loading) (15 mL, 0.15 mmol) was washed with five portions of cold isopropanol then added to a solution of aniline (0.01 mL, 0.11 μmol) and triethylamine (0.2 mL, 1.46 mmol) in dimethyl sulfoxide (6 mL). .. The resulting suspension was vortexed for 24 h, at which point LCMS indicated that the free amine had been consumed.

Enzyme-linked Immunosorbent Assay:

Article Title: Antibodies specific for Epstein-Barr virus nuclear antigen-1 cross-react with human heterogeneous nuclear ribonucleoprotein L
Article Snippet: The EBNA-1 protein was coupled to AffiGel 10 (BioRad, Hercules, CA) according to the manufacturer’s instructions. .. The EBNA-1 protein was coupled to AffiGel 10 (BioRad, Hercules, CA) according to the manufacturer’s instructions.

Article Title: Somatic copy number changes in DPYD are associated with lower risk of recurrence in triple-negative breast cancers
Article Snippet: Anti-DPD antibodies were affinity-purified by coupling the immunogen preparation to a mixture of 50%/50% AffiGel-10 and AffiGel-15 (BioRad, München, Germany). .. Anti-DPD antibodies were affinity-purified by coupling the immunogen preparation to a mixture of 50%/50% AffiGel-10 and AffiGel-15 (BioRad, München, Germany).

Antibody Purification:

Article Title: Phosphoserine aminotransferase 1 is associated to poor outcome on tamoxifen therapy in recurrent breast cancer
Article Snippet: For antibody purification, immunoaffinity columns were prepared using recombinant human full length PSAT1 (including exon 8). .. PSAT1-linked column was prepared by mixing ~2 mL of AffiGel-10® and AffiGel-15 (BioRad) in a 1:1 ratio.

Article Title: The Ndc80 complex targets Bod1 to human mitotic kinetochores
Article Snippet: To prepare the peptide-coated columns, 5 ml Affigel-10 (Bio-Rad) were activated by consecutive treatment with 5% ethylene diamine and 7 mg IAA-NHS ester. .. To prepare the peptide-coated columns, 5 ml Affigel-10 (Bio-Rad) were activated by consecutive treatment with 5% ethylene diamine and 7 mg IAA-NHS ester.

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.

Article Title: Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA
Article Snippet: The coupling reaction was performed by addition of 4.7 mg of His-Pol IV to 0.5 ml AffiGel 10 (Bio-Rad) and mixing for 4 h, followed by a blocking reaction in 20 mM ethanolamine-HCl for 1 h. Pol IV-crosslinked beads were packed into a column (0.5 ml) and were equilibrated in Buffer A (50 mM HEPES–NaOH pH 7.4, 100 mM KCl, 5% glycerol and 1 mM dithiothreitol) as described ( ). .. The coupling reaction was performed by addition of 4.7 mg of His-Pol IV to 0.5 ml AffiGel 10 (Bio-Rad) and mixing for 4 h, followed by a blocking reaction in 20 mM ethanolamine-HCl for 1 h. Pol IV-crosslinked beads were packed into a column (0.5 ml) and were equilibrated in Buffer A (50 mM HEPES–NaOH pH 7.4, 100 mM KCl, 5% glycerol and 1 mM dithiothreitol) as described ( ).

Western Blot:

Article Title: Cdk1 phosphorylation of Esp1/Separase functions with PP2A and Slk19 to regulate pericentric Cohesin and anaphase onset
Article Snippet: Paragraph title: Western blots and immunoprecipitation ... Rabbit serum was harvested, α-GST antibodies were removed on an Affigel-10 (Bio-rad) column coupled to GST, and the α-Slk19 antibodies were purified on an Affigel-10 (Bio-rad) column coupled to purified GST-Slk19700–817 .

Article Title: A Wee1 checkpoint inhibits anaphase onset
Article Snippet: Paragraph title: Western blots and immunoprecipitation ... Plasmids GST-Swe12-312 (in pGEX-4T3, pAR622), malE-Swe12-819 (in pMAL-c2, pAR623), and His6 -Cdk12-298 (pAR727; gifts of Vu Thai and Doug Kellogg, UCSC, Santa Cruz, Santa Cruz, CA; ) were used to produce GST-Swe1 and His6 -Cdk1 protein for injection into rabbits as described above. α-Swe1 antibodies were purified on Affigel-10 (Bio-Rad Laboratories) columns coupled to purified malE-Swe1. α-Cdk1 antibodies were purified using an Affigel-15 (Bio-Rad Laboratories) column coupled to purified His6 -Cdk1, which is insoluble, so the coupling was done in the presence of 0.3% SDS. α-GFP serum (a gift of Aaron Straight [Stanford University, Stanford, CA] and Andrew Murray [Harvard University, Cambridge, MA]) was generated by immunizing rabbits with bacterially expressed His6 -GFP (pAFS97), and purified as above using an Affigel-10 (Bio-Rad Laboratories) column coupled to purified His6 -GFP. α-Cdc20 antibody was generated as described previously ( ).

Article Title: Antibodies specific for Epstein-Barr virus nuclear antigen-1 cross-react with human heterogeneous nuclear ribonucleoprotein L
Article Snippet: The EBNA-1 protein was coupled to AffiGel 10 (BioRad, Hercules, CA) according to the manufacturer’s instructions. .. The EBNA-1 protein was coupled to AffiGel 10 (BioRad, Hercules, CA) according to the manufacturer’s instructions.

Article Title: Somatic copy number changes in DPYD are associated with lower risk of recurrence in triple-negative breast cancers
Article Snippet: Anti-DPD antibodies were affinity-purified by coupling the immunogen preparation to a mixture of 50%/50% AffiGel-10 and AffiGel-15 (BioRad, München, Germany). .. All batches were checked by one-site ELISA using DPD coated on microplates.

Conjugation Assay:

Article Title: Phosphoserine aminotransferase 1 is associated to poor outcome on tamoxifen therapy in recurrent breast cancer
Article Snippet: Peptide synthesis, conjugation to KLH, and rabbit immunization steps were performed by Pineda Antibody Service (Berlin). .. PSAT1-linked column was prepared by mixing ~2 mL of AffiGel-10® and AffiGel-15 (BioRad) in a 1:1 ratio.

Incubation:

Article Title: Engineering streptokinase for generation of active site-labeled plasminogen analogs
Article Snippet: For labeling, 150 μM of 5-(iodoacetamido)fluorescein (5-IAF) was added to 6–15 μM SKΔ(R253-L260)ΔK414-His6 ·ATA-FFR-Pg* complex in the above pH 7.0 buffer and the reaction was initiated by addition of 1 M NH2 OH to a final concentration of 0.1 M. After incubation at 25 °C for 1 h, 50 μM D-Phe-Phe-Arg-CH2 Cl (FFR-CH2 Cl) was added. .. An additional affinity chromatography step using native SK immobilized on AffiGel-10 (Bio-Rad) (1 cm × 16 cm; 5 mg coupled/ml of gel) separated labeled Pg from the SK·Pg/Pm complexes and labeled Pm.

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: Phosphoserine aminotransferase 1 is associated to poor outcome on tamoxifen therapy in recurrent breast cancer
Article Snippet: PSAT1-linked column was prepared by mixing ~2 mL of AffiGel-10® and AffiGel-15 (BioRad) in a 1:1 ratio. .. PSAT1-linked column was prepared by mixing ~2 mL of AffiGel-10® and AffiGel-15 (BioRad) in a 1:1 ratio.

Chromatography:

Article Title: Somatic copy number changes in DPYD are associated with lower risk of recurrence in triple-negative breast cancers
Article Snippet: The recombinant protein was expressed in Escherichia coli cells and subsequently purified by nickel-nitrilotriacetic acid sepharose chromatography (Qiagen) according to standard procedures. .. Anti-DPD antibodies were affinity-purified by coupling the immunogen preparation to a mixture of 50%/50% AffiGel-10 and AffiGel-15 (BioRad, München, Germany).

Concentration Assay:

Article Title: Engineering streptokinase for generation of active site-labeled plasminogen analogs
Article Snippet: For labeling, 150 μM of 5-(iodoacetamido)fluorescein (5-IAF) was added to 6–15 μM SKΔ(R253-L260)ΔK414-His6 ·ATA-FFR-Pg* complex in the above pH 7.0 buffer and the reaction was initiated by addition of 1 M NH2 OH to a final concentration of 0.1 M. After incubation at 25 °C for 1 h, 50 μM D-Phe-Phe-Arg-CH2 Cl (FFR-CH2 Cl) was added. .. An additional affinity chromatography step using native SK immobilized on AffiGel-10 (Bio-Rad) (1 cm × 16 cm; 5 mg coupled/ml of gel) separated labeled Pg from the SK·Pg/Pm complexes and labeled Pm.

Infection:

Article Title: Acetylation of histone H4 lysine 5 and 12 is required for CENP-A deposition into centromeres
Article Snippet: Purified GST fusions were then coupled to Affigel-10 (Bio-Rad) at a ratio of 3.5 mg protein per ml of resin to generate affinity columns. .. Purified GST fusions were then coupled to Affigel-10 (Bio-Rad) at a ratio of 3.5 mg protein per ml of resin to generate affinity columns.

Generated:

Article Title: Cdk1 phosphorylation of Esp1/Separase functions with PP2A and Slk19 to regulate pericentric Cohesin and anaphase onset
Article Snippet: Rabbit serum was harvested, and the α-Mcd1 antibodies purified on an Affigel-10 (Bio-rad) column coupled to purified malE-Mcd1201–301 . malE-Mcd1201–301 was expressed from the plasmid pAR1117 which contains Mcd1201–301 cloned as a BamH1/Sal1 fragment into pMAL-c2 (NEB). α-Slk19 antibodies were generated as follow: coding sequence for the truncated protein Slk19700–817 was amplified by PCR and cloned into pGEX6P-1 (Promega) as a BamHI/EcoRI fragment to create pAR1230. .. Rabbit serum was harvested, α-GST antibodies were removed on an Affigel-10 (Bio-rad) column coupled to GST, and the α-Slk19 antibodies were purified on an Affigel-10 (Bio-rad) column coupled to purified GST-Slk19700–817 .

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. .. The GST-Dia2 protein was coupled to Affigel-10 (Bio-Rad) according to the manufacturer's instructions.

Article Title: A Wee1 checkpoint inhibits anaphase onset
Article Snippet: The triethylamine and glycine elutions were neutralized, dialyzed in PBS + 50% glycerol, and stored at −80°C. .. Plasmids GST-Swe12-312 (in pGEX-4T3, pAR622), malE-Swe12-819 (in pMAL-c2, pAR623), and His6 -Cdk12-298 (pAR727; gifts of Vu Thai and Doug Kellogg, UCSC, Santa Cruz, Santa Cruz, CA; ) were used to produce GST-Swe1 and His6 -Cdk1 protein for injection into rabbits as described above. α-Swe1 antibodies were purified on Affigel-10 (Bio-Rad Laboratories) columns coupled to purified malE-Swe1. α-Cdk1 antibodies were purified using an Affigel-15 (Bio-Rad Laboratories) column coupled to purified His6 -Cdk1, which is insoluble, so the coupling was done in the presence of 0.3% SDS. α-GFP serum (a gift of Aaron Straight [Stanford University, Stanford, CA] and Andrew Murray [Harvard University, Cambridge, MA]) was generated by immunizing rabbits with bacterially expressed His6 -GFP (pAFS97), and purified as above using an Affigel-10 (Bio-Rad Laboratories) column coupled to purified His6 -GFP. α-Cdc20 antibody was generated as described previously ( ). .. Rabbit sera was purified as above using an Affigel-15 (Bio-Rad Laboratories) column coupled to purified His6 -Cdc20470-610 , which was expressed from a BamH1–Not1 fragment cloned into pHIS-parallel2 to create pAR784.

Article Title: An Sp1 transcription factor coordinates caspase-dependent and -independent apoptotic pathways
Article Snippet: Polyclonal antibodies were affinity-purified using identical SPTF-3 fragments fused to maltose-binding protein (MBP) and coupled to Affigel 10 (Bio-Rad). .. Polyclonal antibodies were affinity-purified using identical SPTF-3 fragments fused to maltose-binding protein (MBP) and coupled to Affigel 10 (Bio-Rad).

Article Title: Acetylation of histone H4 lysine 5 and 12 is required for CENP-A deposition into centromeres
Article Snippet: Purified GST fusions were then coupled to Affigel-10 (Bio-Rad) at a ratio of 3.5 mg protein per ml of resin to generate affinity columns. .. Purified GST fusions were then coupled to Affigel-10 (Bio-Rad) at a ratio of 3.5 mg protein per ml of resin to generate affinity columns.

Imaging:

Article Title: Non-glycanated Decorin Is a Drug Target on Human Adipose Stromal Cells
Article Snippet: Six milligrams of a cysteine-cyclized peptide was coupled onto 250 μL of Affigel 10 (Bio-Rad), and the column was equilibrated with column buffer containing 1% Triton X-100. .. Protein preparations resolved on SDS-PAGE were blotted onto Immobilon-FL membrane (Millipore), blocked with Odyssey blocking buffer (LI-COR Biosciences), and probed (in PBS/0.05% Triton X-100) with 1:1,000 goat anti-DCN (R & D Systems).

Polymerase Chain Reaction:

Article Title: Cdk1 phosphorylation of Esp1/Separase functions with PP2A and Slk19 to regulate pericentric Cohesin and anaphase onset
Article Snippet: Rabbit serum was harvested, and the α-Mcd1 antibodies purified on an Affigel-10 (Bio-rad) column coupled to purified malE-Mcd1201–301 . malE-Mcd1201–301 was expressed from the plasmid pAR1117 which contains Mcd1201–301 cloned as a BamH1/Sal1 fragment into pMAL-c2 (NEB). α-Slk19 antibodies were generated as follow: coding sequence for the truncated protein Slk19700–817 was amplified by PCR and cloned into pGEX6P-1 (Promega) as a BamHI/EcoRI fragment to create pAR1230. .. Rabbit serum was harvested, α-GST antibodies were removed on an Affigel-10 (Bio-rad) column coupled to GST, and the α-Slk19 antibodies were purified on an Affigel-10 (Bio-rad) column coupled to purified GST-Slk19700–817 .

Article Title: A Wee1 checkpoint inhibits anaphase onset
Article Snippet: Coding sequences for the truncated proteins Clb52-137 , Pds1178-373 , and Sgo1129-326 were amplified using PCR and cloned into pGEX6P-1 (Promega) as BamH1–EcoR1 fragments to create pAR627, pAR624, and pAR717, respectively. .. Plasmids GST-Swe12-312 (in pGEX-4T3, pAR622), malE-Swe12-819 (in pMAL-c2, pAR623), and His6 -Cdk12-298 (pAR727; gifts of Vu Thai and Doug Kellogg, UCSC, Santa Cruz, Santa Cruz, CA; ) were used to produce GST-Swe1 and His6 -Cdk1 protein for injection into rabbits as described above. α-Swe1 antibodies were purified on Affigel-10 (Bio-Rad Laboratories) columns coupled to purified malE-Swe1. α-Cdk1 antibodies were purified using an Affigel-15 (Bio-Rad Laboratories) column coupled to purified His6 -Cdk1, which is insoluble, so the coupling was done in the presence of 0.3% SDS. α-GFP serum (a gift of Aaron Straight [Stanford University, Stanford, CA] and Andrew Murray [Harvard University, Cambridge, MA]) was generated by immunizing rabbits with bacterially expressed His6 -GFP (pAFS97), and purified as above using an Affigel-10 (Bio-Rad Laboratories) column coupled to purified His6 -GFP. α-Cdc20 antibody was generated as described previously ( ).

Injection:

Article Title: Cdk1 phosphorylation of Esp1/Separase functions with PP2A and Slk19 to regulate pericentric Cohesin and anaphase onset
Article Snippet: GST-Slk19700–817 was purified and 1 mg of the fusion protein was injected into rabbits every 4 weeks for 8 to 16 weeks (uOttawa animal facility). .. Rabbit serum was harvested, α-GST antibodies were removed on an Affigel-10 (Bio-rad) column coupled to GST, and the α-Slk19 antibodies were purified on an Affigel-10 (Bio-rad) column coupled to purified GST-Slk19700–817 .

Article Title: A Wee1 checkpoint inhibits anaphase onset
Article Snippet: The triethylamine and glycine elutions were neutralized, dialyzed in PBS + 50% glycerol, and stored at −80°C. .. Plasmids GST-Swe12-312 (in pGEX-4T3, pAR622), malE-Swe12-819 (in pMAL-c2, pAR623), and His6 -Cdk12-298 (pAR727; gifts of Vu Thai and Doug Kellogg, UCSC, Santa Cruz, Santa Cruz, CA; ) were used to produce GST-Swe1 and His6 -Cdk1 protein for injection into rabbits as described above. α-Swe1 antibodies were purified on Affigel-10 (Bio-Rad Laboratories) columns coupled to purified malE-Swe1. α-Cdk1 antibodies were purified using an Affigel-15 (Bio-Rad Laboratories) column coupled to purified His6 -Cdk1, which is insoluble, so the coupling was done in the presence of 0.3% SDS. α-GFP serum (a gift of Aaron Straight [Stanford University, Stanford, CA] and Andrew Murray [Harvard University, Cambridge, MA]) was generated by immunizing rabbits with bacterially expressed His6 -GFP (pAFS97), and purified as above using an Affigel-10 (Bio-Rad Laboratories) column coupled to purified His6 -GFP. α-Cdc20 antibody was generated as described previously ( ). .. Rabbit sera was purified as above using an Affigel-15 (Bio-Rad Laboratories) column coupled to purified His6 -Cdc20470-610 , which was expressed from a BamH1–Not1 fragment cloned into pHIS-parallel2 to create pAR784.

Article Title: Phosphoserine aminotransferase 1 is associated to poor outcome on tamoxifen therapy in recurrent breast cancer
Article Snippet: KLH-conjugated peptides were injected into rabbits, which received boost immunizations at day 20, 30, 40, 61, 75, 90, and 104. .. PSAT1-linked column was prepared by mixing ~2 mL of AffiGel-10® and AffiGel-15 (BioRad) in a 1:1 ratio.

Article Title: Cep169, a Novel Microtubule Plus-End-Tracking Centrosomal Protein, Binds to CDK5RAP2 and Regulates Microtubule Stability
Article Snippet: The purified protein was injected into rabbits to raise polyclonal anti-Cep169 antisera. .. The resulting sera were further purified using GST-Cep169 (1–100) immobilized with Affigel-10 (Bio-Rad Laboratories).

Article Title: The Ndc80 complex targets Bod1 to human mitotic kinetochores
Article Snippet: To prepare the peptide-coated columns, 5 ml Affigel-10 (Bio-Rad) were activated by consecutive treatment with 5% ethylene diamine and 7 mg IAA-NHS ester. .. To prepare the peptide-coated columns, 5 ml Affigel-10 (Bio-Rad) were activated by consecutive treatment with 5% ethylene diamine and 7 mg IAA-NHS ester.

Recombinant:

Article Title: Phosphoserine aminotransferase 1 is associated to poor outcome on tamoxifen therapy in recurrent breast cancer
Article Snippet: Recombinant PSAT1 was expressed in E. coli as a N-terminal histidine tagged protein and was purified via Ni2+ -NTA (Qiagen) affinity chromatography under denaturing and slightly reduced conditions (purity > 95%). .. PSAT1-linked column was prepared by mixing ~2 mL of AffiGel-10® and AffiGel-15 (BioRad) in a 1:1 ratio.

Article Title: Somatic copy number changes in DPYD are associated with lower risk of recurrence in triple-negative breast cancers
Article Snippet: The recombinant protein was expressed in Escherichia coli cells and subsequently purified by nickel-nitrilotriacetic acid sepharose chromatography (Qiagen) according to standard procedures. .. Anti-DPD antibodies were affinity-purified by coupling the immunogen preparation to a mixture of 50%/50% AffiGel-10 and AffiGel-15 (BioRad, München, Germany).

Pull Down Assay:

Article Title: Non-glycanated Decorin Is a Drug Target on Human Adipose Stromal Cells
Article Snippet: Protein pull-down assay was performed as described. .. Six milligrams of a cysteine-cyclized peptide was coupled onto 250 μL of Affigel 10 (Bio-Rad), and the column was equilibrated with column buffer containing 1% Triton X-100.

Labeling:

Article Title: Engineering streptokinase for generation of active site-labeled plasminogen analogs
Article Snippet: Most of SKΔ(R253-L260)ΔK414-His6 remained bound to the Ni2+ -Sepharose column and was eluted with 500 mM imidazole in 50 mM Hepes, 0.3 M NaCl, pH 7.4. .. An additional affinity chromatography step using native SK immobilized on AffiGel-10 (Bio-Rad) (1 cm × 16 cm; 5 mg coupled/ml of gel) separated labeled Pg from the SK·Pg/Pm complexes and labeled Pm. .. The complexes were eluted with 0.1 M Hepes, 0.125 M NaCl, 1 mM EDTA, 10 mM 6-AHA, pH 7.4 buffer.

Purification:

Article Title: Cdk1 phosphorylation of Esp1/Separase functions with PP2A and Slk19 to regulate pericentric Cohesin and anaphase onset
Article Snippet: GST-Slk19700–817 was purified and 1 mg of the fusion protein was injected into rabbits every 4 weeks for 8 to 16 weeks (uOttawa animal facility). .. Rabbit serum was harvested, α-GST antibodies were removed on an Affigel-10 (Bio-rad) column coupled to GST, and the α-Slk19 antibodies were purified on an Affigel-10 (Bio-rad) column coupled to purified GST-Slk19700–817 . .. HRP-conjugated α-rabbit and α-mouse secondary antibodies (Bio-rad) were used at a 1:5000 dilution in TBS-T + 4% Fat Free Milk Powder for 30 min to 1 hr., washed with TBS-T and incubated in Western Lightning Plus-ECL (PerkinElmer).

Article Title: Prefoldin-Nascent Chain Complexes in the Folding of Cytoskeletal Proteins
Article Snippet: A purified rat mAb, 23C, recognizing TCP-1α, was used for the analysis of CCT ( ). .. Purified rabbit skeletal muscle actin was obtained from Cytoskeleton Inc. AffiGel-10 was from Bio-Rad. .. DNase I was from Worthington Biochemicals.

Article Title: A Wee1 checkpoint inhibits anaphase onset
Article Snippet: The triethylamine and glycine elutions were neutralized, dialyzed in PBS + 50% glycerol, and stored at −80°C. .. Plasmids GST-Swe12-312 (in pGEX-4T3, pAR622), malE-Swe12-819 (in pMAL-c2, pAR623), and His6 -Cdk12-298 (pAR727; gifts of Vu Thai and Doug Kellogg, UCSC, Santa Cruz, Santa Cruz, CA; ) were used to produce GST-Swe1 and His6 -Cdk1 protein for injection into rabbits as described above. α-Swe1 antibodies were purified on Affigel-10 (Bio-Rad Laboratories) columns coupled to purified malE-Swe1. α-Cdk1 antibodies were purified using an Affigel-15 (Bio-Rad Laboratories) column coupled to purified His6 -Cdk1, which is insoluble, so the coupling was done in the presence of 0.3% SDS. α-GFP serum (a gift of Aaron Straight [Stanford University, Stanford, CA] and Andrew Murray [Harvard University, Cambridge, MA]) was generated by immunizing rabbits with bacterially expressed His6 -GFP (pAFS97), and purified as above using an Affigel-10 (Bio-Rad Laboratories) column coupled to purified His6 -GFP. α-Cdc20 antibody was generated as described previously ( ). .. Rabbit sera was purified as above using an Affigel-15 (Bio-Rad Laboratories) column coupled to purified His6 -Cdc20470-610 , which was expressed from a BamH1–Not1 fragment cloned into pHIS-parallel2 to create pAR784.

Article Title: An Sp1 transcription factor coordinates caspase-dependent and -independent apoptotic pathways
Article Snippet: Protein fragments corresponding to amino acids 1–79 and 192–275 of SPTF-3 fused to glutathione S- transferase (GST) were expressed, purified using glutathione Sepharose 4B (Amersham Biosciences) and used to raise SPTF-3 N81 or SPTF-3 M82 antibodies, respectively. .. Polyclonal antibodies were affinity-purified using identical SPTF-3 fragments fused to maltose-binding protein (MBP) and coupled to Affigel 10 (Bio-Rad).

Article Title: Segregation of molecules at cell division reveals native protein localization
Article Snippet: The Alexa 350 fluorophores were imaged with a DAPI (LF405-A, Semrock) filter cube. .. Antibodies to ClpX were purified using Affigel-10 (Bio-Rad) resin. .. The E. coli ClpX protein was purified according to previous protocols , conjugated to resin, and ClpX polyclonal rabbit antibodies (Covance) were purified according to the manufacture's protocol.

Article Title: Acetylation of histone H4 lysine 5 and 12 is required for CENP-A deposition into centromeres
Article Snippet: Peak fractions were pooled and dialyzed into 10 mM HEPES pH 8.0, 500 mM NaCl and 10% glycerol. .. Purified GST fusions were then coupled to Affigel-10 (Bio-Rad) at a ratio of 3.5 mg protein per ml of resin to generate affinity columns. .. For histone tail binding experiments, purified GST fusions were coupled to NHS-Sepharose (GE-Healthcare) at a ratio of 5 mg protein per ml of resin.

Article Title: Phosphoserine aminotransferase 1 is associated to poor outcome on tamoxifen therapy in recurrent breast cancer
Article Snippet: Recombinant PSAT1 was expressed in E. coli as a N-terminal histidine tagged protein and was purified via Ni2+ -NTA (Qiagen) affinity chromatography under denaturing and slightly reduced conditions (purity > 95%). .. PSAT1-linked column was prepared by mixing ~2 mL of AffiGel-10® and AffiGel-15 (BioRad) in a 1:1 ratio.

Article Title: Cep169, a Novel Microtubule Plus-End-Tracking Centrosomal Protein, Binds to CDK5RAP2 and Regulates Microtubule Stability
Article Snippet: To affinity purify the anti-Cep169 antibody, immunized sera were first depleted of anti-GST antibody using a GST column. .. The resulting sera were further purified using GST-Cep169 (1–100) immobilized with Affigel-10 (Bio-Rad Laboratories). .. 293T cells were transfected with EGFP-Cep169 and FLAG-CDK5RAP2 and extracted by extraction buffer (20 mM Tris-HCl at pH 7.5, 100 mM NaCl, 5 mM MgCl2 , 0.2% Nonidet P-40, 10% glycerol, 1 mM NaF, 1 mM Na3 VO4 , 20 mM β-glycerophosphate, 10 mM β-mercaptoethanol, 0.2 mM PMSF, Protease Inhibitor Cocktail [Nacarai Tesque]) by two rounds of freezing and thawing as described previously [ ].

Article Title: Antibodies specific for Epstein-Barr virus nuclear antigen-1 cross-react with human heterogeneous nuclear ribonucleoprotein L
Article Snippet: The EBNA-1 protein was coupled to AffiGel 10 (BioRad, Hercules, CA) according to the manufacturer’s instructions. .. The EBNA-1 protein was coupled to AffiGel 10 (BioRad, Hercules, CA) according to the manufacturer’s instructions.

Article Title: Somatic copy number changes in DPYD are associated with lower risk of recurrence in triple-negative breast cancers
Article Snippet: The recombinant protein was expressed in Escherichia coli cells and subsequently purified by nickel-nitrilotriacetic acid sepharose chromatography (Qiagen) according to standard procedures. .. Anti-DPD antibodies were affinity-purified by coupling the immunogen preparation to a mixture of 50%/50% AffiGel-10 and AffiGel-15 (BioRad, München, Germany).

Article Title: The Ndc80 complex targets Bod1 to human mitotic kinetochores
Article Snippet: The total Bod1 antibody was purified using only the non-phosphopeptide column. .. To prepare the peptide-coated columns, 5 ml Affigel-10 (Bio-Rad) were activated by consecutive treatment with 5% ethylene diamine and 7 mg IAA-NHS ester.

Article Title: Visualizing and Analyzing Branching Microtubule Nucleation Using Meiotic Xenopus Egg Extracts and TIRF Microscopy
Article Snippet: Paragraph title: 2.1.3 Rabbit Polyclonal Antibody Production and Purification for Immunodepletion ... Affigel-10 and -15 matrix (Bio-Rad).

Article Title: Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA
Article Snippet: Purified His-Pol IV was dialyzed against 100 mM HEPES–NaOH pH 7.4 and 200 mM NaCl at 4°C. .. The coupling reaction was performed by addition of 4.7 mg of His-Pol IV to 0.5 ml AffiGel 10 (Bio-Rad) and mixing for 4 h, followed by a blocking reaction in 20 mM ethanolamine-HCl for 1 h. Pol IV-crosslinked beads were packed into a column (0.5 ml) and were equilibrated in Buffer A (50 mM HEPES–NaOH pH 7.4, 100 mM KCl, 5% glycerol and 1 mM dithiothreitol) as described ( ).

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.

Sequencing:

Article Title: Cdk1 phosphorylation of Esp1/Separase functions with PP2A and Slk19 to regulate pericentric Cohesin and anaphase onset
Article Snippet: Rabbit serum was harvested, and the α-Mcd1 antibodies purified on an Affigel-10 (Bio-rad) column coupled to purified malE-Mcd1201–301 . malE-Mcd1201–301 was expressed from the plasmid pAR1117 which contains Mcd1201–301 cloned as a BamH1/Sal1 fragment into pMAL-c2 (NEB). α-Slk19 antibodies were generated as follow: coding sequence for the truncated protein Slk19700–817 was amplified by PCR and cloned into pGEX6P-1 (Promega) as a BamHI/EcoRI fragment to create pAR1230. .. Rabbit serum was harvested, α-GST antibodies were removed on an Affigel-10 (Bio-rad) column coupled to GST, and the α-Slk19 antibodies were purified on an Affigel-10 (Bio-rad) column coupled to purified GST-Slk19700–817 .

Silver Staining:

Article Title: Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA
Article Snippet: The coupling reaction was performed by addition of 4.7 mg of His-Pol IV to 0.5 ml AffiGel 10 (Bio-Rad) and mixing for 4 h, followed by a blocking reaction in 20 mM ethanolamine-HCl for 1 h. Pol IV-crosslinked beads were packed into a column (0.5 ml) and were equilibrated in Buffer A (50 mM HEPES–NaOH pH 7.4, 100 mM KCl, 5% glycerol and 1 mM dithiothreitol) as described ( ). .. The coupling reaction was performed by addition of 4.7 mg of His-Pol IV to 0.5 ml AffiGel 10 (Bio-Rad) and mixing for 4 h, followed by a blocking reaction in 20 mM ethanolamine-HCl for 1 h. Pol IV-crosslinked beads were packed into a column (0.5 ml) and were equilibrated in Buffer A (50 mM HEPES–NaOH pH 7.4, 100 mM KCl, 5% glycerol and 1 mM dithiothreitol) as described ( ).

SDS Page:

Article Title: Non-glycanated Decorin Is a Drug Target on Human Adipose Stromal Cells
Article Snippet: Six milligrams of a cysteine-cyclized peptide was coupled onto 250 μL of Affigel 10 (Bio-Rad), and the column was equilibrated with column buffer containing 1% Triton X-100. .. Peptide-coupled resin (10 μL) was incubated with 30 μg of membrane extract and washed with column buffer.

Plasmid Preparation:

Article Title: Cdk1 phosphorylation of Esp1/Separase functions with PP2A and Slk19 to regulate pericentric Cohesin and anaphase onset
Article Snippet: Rabbit serum was harvested, and the α-Mcd1 antibodies purified on an Affigel-10 (Bio-rad) column coupled to purified malE-Mcd1201–301 . malE-Mcd1201–301 was expressed from the plasmid pAR1117 which contains Mcd1201–301 cloned as a BamH1/Sal1 fragment into pMAL-c2 (NEB). α-Slk19 antibodies were generated as follow: coding sequence for the truncated protein Slk19700–817 was amplified by PCR and cloned into pGEX6P-1 (Promega) as a BamHI/EcoRI fragment to create pAR1230. .. Rabbit serum was harvested, α-GST antibodies were removed on an Affigel-10 (Bio-rad) column coupled to GST, and the α-Slk19 antibodies were purified on an Affigel-10 (Bio-rad) column coupled to purified GST-Slk19700–817 .

Article Title: Somatic copy number changes in DPYD are associated with lower risk of recurrence in triple-negative breast cancers
Article Snippet: Human DPYD cDNA was cloned into the Nco I/Bgl II restriction sites of a pQE-60 vector (Qiagen) including a polyhistidine (His6 )-tag at the C-terminus. .. Anti-DPD antibodies were affinity-purified by coupling the immunogen preparation to a mixture of 50%/50% AffiGel-10 and AffiGel-15 (BioRad, München, Germany).

Affinity Chromatography:

Article Title: Engineering streptokinase for generation of active site-labeled plasminogen analogs
Article Snippet: Most of SKΔ(R253-L260)ΔK414-His6 remained bound to the Ni2+ -Sepharose column and was eluted with 500 mM imidazole in 50 mM Hepes, 0.3 M NaCl, pH 7.4. .. An additional affinity chromatography step using native SK immobilized on AffiGel-10 (Bio-Rad) (1 cm × 16 cm; 5 mg coupled/ml of gel) separated labeled Pg from the SK·Pg/Pm complexes and labeled Pm. .. The complexes were eluted with 0.1 M Hepes, 0.125 M NaCl, 1 mM EDTA, 10 mM 6-AHA, pH 7.4 buffer.

Article Title: Non-glycanated Decorin Is a Drug Target on Human Adipose Stromal Cells
Article Snippet: Paragraph title: Affinity Chromatography ... Six milligrams of a cysteine-cyclized peptide was coupled onto 250 μL of Affigel 10 (Bio-Rad), and the column was equilibrated with column buffer containing 1% Triton X-100.

Article Title: Phosphoserine aminotransferase 1 is associated to poor outcome on tamoxifen therapy in recurrent breast cancer
Article Snippet: Recombinant PSAT1 was expressed in E. coli as a N-terminal histidine tagged protein and was purified via Ni2+ -NTA (Qiagen) affinity chromatography under denaturing and slightly reduced conditions (purity > 95%). .. PSAT1-linked column was prepared by mixing ~2 mL of AffiGel-10® and AffiGel-15 (BioRad) in a 1:1 ratio.

Column Chromatography:

Article Title: Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA
Article Snippet: Paragraph title: Pol IV-affinity column chromatography ... The coupling reaction was performed by addition of 4.7 mg of His-Pol IV to 0.5 ml AffiGel 10 (Bio-Rad) and mixing for 4 h, followed by a blocking reaction in 20 mM ethanolamine-HCl for 1 h. Pol IV-crosslinked beads were packed into a column (0.5 ml) and were equilibrated in Buffer A (50 mM HEPES–NaOH pH 7.4, 100 mM KCl, 5% glycerol and 1 mM dithiothreitol) as described ( ).

Quantitation Assay:

Article Title: Engineering streptokinase for generation of active site-labeled plasminogen analogs
Article Snippet: An additional affinity chromatography step using native SK immobilized on AffiGel-10 (Bio-Rad) (1 cm × 16 cm; 5 mg coupled/ml of gel) separated labeled Pg from the SK·Pg/Pm complexes and labeled Pm. .. Labeled Pg was concentrated with a YM-30 ultrafiltration membrane and dialyzed against > 5000 volumes of 50 mM Hepes, 125 mM NaCl, 1 mM EDTA, 1 mg/ml PEG 8000, pH 7.4.

Produced:

Article Title: Cep169, a Novel Microtubule Plus-End-Tracking Centrosomal Protein, Binds to CDK5RAP2 and Regulates Microtubule Stability
Article Snippet: Cep169 (1–100) [“(1–100)” indicates amino acids 1 to 100] was produced as glutathione S-transferase (GST) fusion protein in E . coli and purified using GST column (Amersham Biosciences) as described previously [ ]. .. The resulting sera were further purified using GST-Cep169 (1–100) immobilized with Affigel-10 (Bio-Rad Laboratories).

Immunoprecipitation:

Article Title: Cdk1 phosphorylation of Esp1/Separase functions with PP2A and Slk19 to regulate pericentric Cohesin and anaphase onset
Article Snippet: Paragraph title: Western blots and immunoprecipitation ... Rabbit serum was harvested, α-GST antibodies were removed on an Affigel-10 (Bio-rad) column coupled to GST, and the α-Slk19 antibodies were purified on an Affigel-10 (Bio-rad) column coupled to purified GST-Slk19700–817 .

Article Title: A Wee1 checkpoint inhibits anaphase onset
Article Snippet: Paragraph title: Western blots and immunoprecipitation ... Plasmids GST-Swe12-312 (in pGEX-4T3, pAR622), malE-Swe12-819 (in pMAL-c2, pAR623), and His6 -Cdk12-298 (pAR727; gifts of Vu Thai and Doug Kellogg, UCSC, Santa Cruz, Santa Cruz, CA; ) were used to produce GST-Swe1 and His6 -Cdk1 protein for injection into rabbits as described above. α-Swe1 antibodies were purified on Affigel-10 (Bio-Rad Laboratories) columns coupled to purified malE-Swe1. α-Cdk1 antibodies were purified using an Affigel-15 (Bio-Rad Laboratories) column coupled to purified His6 -Cdk1, which is insoluble, so the coupling was done in the presence of 0.3% SDS. α-GFP serum (a gift of Aaron Straight [Stanford University, Stanford, CA] and Andrew Murray [Harvard University, Cambridge, MA]) was generated by immunizing rabbits with bacterially expressed His6 -GFP (pAFS97), and purified as above using an Affigel-10 (Bio-Rad Laboratories) column coupled to purified His6 -GFP. α-Cdc20 antibody was generated as described previously ( ).

BAC Assay:

Article Title: Acetylation of histone H4 lysine 5 and 12 is required for CENP-A deposition into centromeres
Article Snippet: Purified GST fusions were then coupled to Affigel-10 (Bio-Rad) at a ratio of 3.5 mg protein per ml of resin to generate affinity columns. .. For histone tail binding experiments, purified GST fusions were coupled to NHS-Sepharose (GE-Healthcare) at a ratio of 5 mg protein per ml of resin.

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.

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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
    Affigel 10 Slurry Resin, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 77/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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:

    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:

    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

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

    Journal:

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

    doi: 10.1016/j.ab.2011.04.025

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

    Article Snippet: An additional affinity chromatography step using native SK immobilized on AffiGel-10 (Bio-Rad) (1 cm × 16 cm; 5 mg coupled/ml of gel) separated labeled Pg from the SK·Pg/Pm complexes and labeled Pm.

    Techniques: Purification, Labeling, Affinity Chromatography