Extracts from strains WT, ∆nop17, ∆rsa1 and ∆tah1 expressing ProtA-Nop58 that had been grown in galactose medium were prepared in buffer (20 mM Tris-Cl pH 8.0; 0.5 mM magnesium acetate; 0.2% Triton X-100; 150 mM potassium acetate; 1 mM DTT and 1 mM PMSF). Immunoprecipitation was performed by incubating total extract with IgG-Sepharose (Amersham Biosciences) for 2 hours at 4°C. Fractions corresponding to total extract (TE), flow-trough (FT), wash (W) and immunoprecipitation (IP) were collected and stored at −20°C. For protein analysis by western blot, samples were separated by SDS-PAGE and transferred to PVDF membranes (GE Healthcare). For ProtA-Nop58 and HA-Hsc82 expression in WT and ∆nop17, the same experiment strategy was used.
Igg sepharose
Manufactured by Cytiva
Sourced in United States, Japan
IgG Sepharose is a chromatography resin designed for the purification of immunoglobulin G (IgG) from various sample sources. It consists of cross-linked agarose beads to which protein A, a bacterial surface protein with high affinity for the Fc region of IgG, is covalently coupled. IgG Sepharose can be used in affinity chromatography for the capture and purification of IgG from complex mixtures.
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6 protocols using igg sepharose
1
Immunoprecipitation of ProtA-Nop58 in Yeast
2
Isolation and Analysis of Utp21-TAP Complexes
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To isolate Utp21-TAP complexes, utp21::MET2 cells expressing pRS414ADHUTP21 were grown overnight to an OD.600 of ∼2.0. Cells were disrupted in lysis buffer (20 mM Tris, pH 7.5, 100 mM KCl, 5 mM MgCl2, plus a protease inhibitor tablet (Roche Applied Science)) in the presence of glass beads. Yeast lysate was incubated with IgG sepharose (Amersham Biosciences) (1 hour, 4°C) followed by washes with 20 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.1% Tween-20. Proteins bound to IgG sepharose were eluted by boiling in SDS-PAGE sample buffer and separated by gel electrophoresis (7.5% SDS-polyacrylamide gel unless otherwise indicated). Alternatively, cells (0.5 OD.600 units) were resuspended in cold phosphate-buffered saline containing 1 mM phenylmethylsulfonylfluoride and disrupted with glass beads in the presence of SDS and triton X-100. For immunoblot analysis, proteins were transferred to nitrocellulose and probed with indicated antibodies. Chemiluminescence immunoblots were performed according to the manufacturer’s suggestions (Pierce, Rockford, IL). Polyclonal anti-TAP antibodies were obtained from ThermoScientific. Polyclonal antibodies against Hsc82/Hsp82, Sti1 or Cpr6 have been described [34] (link), [57] (link). The antibody against Zuo1 was a gift from Dr. Elizabeth Craig (University of Wisconsin-Madison).
3
Purification of TAP-tagged Protein Complexes
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HEK293 human cells were cultured in Falcon© six-well plates with DMEM supplemented with 5% (vol/vol) FBS. Cells at 80% of confluency were transfected using Lipofectamine LTX (Thermo Fisher Scientific) and were harvested 24 hpt. The complexes associated to the TAP-tagged proteins were purified as described (Francisco-Velilla et al, 2016 (link)). Briefly, the extract from the TEV protease digestion of the first IgG Sepharose (Cytiva) purification was subsequently subjected to a second calmodulin (Agilent Technologies) purification step. Purified proteins were precipitated with 10% trichloroacetic acid at 4°C overnight, pelleted at 14,000 g for 15 min at 4°C, washed three times with 1 ml of acetone, and dissolved in SDS–loading buffer. An aliquot (20%) was analyzed on silver-stained SDS–PAGE gels to visualize the purification of proteins associated to G5845-1508-TAP polypeptides.
4
Chia Protein Chimera Creation
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Mouse and cattle Chia have similar exon structures at the nucleotide level. To create mouse/cattle chimeric proteins, we fused two units at the junctions among exons 3–5, exons 6–7, exons 8–10 and exon 11 using template DNAs and primers (Figure S1 ; Tables S4 and S5 ) as described previously.35 More chimeras were also produced by combining templates and primers (Tables S4 and S5 ). Chia mutant proteins were prepared by PCR using a template and primers (Tables S4 and S5 ).35
E. coli BL21 (DE3) was transformed to express pre-Protein A-Chia-V5-His proteins using the plasmid DNAs. Transformed E. coli were grown in 250 mL of LB medium containing 100 μg/mL ampicillin at 37°C for 18 h. After induction with 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG), the bacteria were cultured for two h in an LB medium. Cells were harvested by centrifugation at 6,500 g for 20 min at 4°C. The recombinant protein was prepared from E. coli and purified by IgG Sepharose (Cytiva, Marlborough, MA, USA) chromatography as described previously.35 The protein-containing fractions were desalted using PD MidiTrap G-25 (Cytiva) equilibrated with TS buffer [20 mM Tris-HCl (pH 7.6), 150 mM NaCl and a protease inhibitor (Complete, Roche, Basel, Switzerland)]. Western blot detected recombinant products using an anti-V5-HRP monoclonal antibody (Thermo Fisher Scientific, Waltham, MA, USA).
E. coli BL21 (DE3) was transformed to express pre-Protein A-Chia-V5-His proteins using the plasmid DNAs. Transformed E. coli were grown in 250 mL of LB medium containing 100 μg/mL ampicillin at 37°C for 18 h. After induction with 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG), the bacteria were cultured for two h in an LB medium. Cells were harvested by centrifugation at 6,500 g for 20 min at 4°C. The recombinant protein was prepared from E. coli and purified by IgG Sepharose (Cytiva, Marlborough, MA, USA) chromatography as described previously.35 The protein-containing fractions were desalted using PD MidiTrap G-25 (Cytiva) equilibrated with TS buffer [20 mM Tris-HCl (pH 7.6), 150 mM NaCl and a protease inhibitor (Complete, Roche, Basel, Switzerland)]. Western blot detected recombinant products using an anti-V5-HRP monoclonal antibody (Thermo Fisher Scientific, Waltham, MA, USA).
5
Recombinant Expression and Kinase Assay of FAM20 Proteins
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The cDNA fragment of the truncated FAM20C sequence lacking the first 42 N-terminal amino acids was PCR-amplified with pCMV-FAM20C as the template using a primer set designed to have BglII sites at each primer (Supplementary Table 8 ). The PCR fragment was then subcloned into the BamHI site of pGIR201proA61 (link), resulting in the fusion of the insulin signal and the protein A sequences present in the vector (pEF-BOS/IP-FAM20C). Soluble FAM20C mutants were constructed by PCR-based site-directed mutagenesis as described above. Resulting plasmid constructs (pEF-BOS/IP-FAM20Cs, D478G, G379R, G379E, L388R, and R549W) were confirmed for fidelity by sequencing. A kinase-dead mutant of FAM20B (D309G) was constructed using site-directed mutagenesis.
The expression vectors (6.0 µg), including pEF-BOS/IP-FAM20B23 (link) and pEF-BOS/IP-FAM20Cs, were individually transfected into COS-1 cells on 100-mm plates using FuGENE™ 6, as described above. For the co-transfection experiments, pEF-BOS/IP-C4ST-162 (link) and each of the FAM20-related expression plasmids (3.0 µg each) were used. Two days after transfection, 1 ml of culture medium was collected and incubated with 10 µl of IgG-sepharose (Cytiva, Tokyo, Japan) for 1 h at 4 °C. The beads were recovered by centrifugation, washed, resuspended in assay buffer, and tested for XYLK and sulfotransferase activities23 (link),62 (link).
The expression vectors (6.0 µg), including pEF-BOS/IP-FAM20B23 (link) and pEF-BOS/IP-FAM20Cs, were individually transfected into COS-1 cells on 100-mm plates using FuGENE™ 6, as described above. For the co-transfection experiments, pEF-BOS/IP-C4ST-162 (link) and each of the FAM20-related expression plasmids (3.0 µg each) were used. Two days after transfection, 1 ml of culture medium was collected and incubated with 10 µl of IgG-sepharose (Cytiva, Tokyo, Japan) for 1 h at 4 °C. The beads were recovered by centrifugation, washed, resuspended in assay buffer, and tested for XYLK and sulfotransferase activities23 (link),62 (link).
6
Tandem Affinity Purification of G5 Protein Complexes
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HEK293 cells were cultured with DMEM supplemented with 5 % fetal calf serum. Monolayers grown at 80 % of confluency were transfected with the constructs G5845-1508-TAP and were harvested 24 h post-treatment (hpt). The complexes associated with the TAP-tagged proteins were purified as described [11] (link). Briefly, the extract from the TEV protease digestion of the first IgG Sepharose (Cytiva) purification was subsequently subjected to a second calmodulin (Agilent Technologies) purification step. Purified proteins were precipitated with 10 % trichloroacetic acid, washed with acetone, and dissolved in SDS–loading buffer. An aliquot (20 %) was analyzed on silver-stained SDS–polyacrylamide (PAGE) gels to visualize the purification of proteins associated with G5845-1508-TAP polypeptides.
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