Capture of HIF-1α interacting proteins from cell lysates was performed as described (28 (link), 29 (link)), with minor modifications. Briefly, GST or GST-HIF-1α531-826 was covalently coupled to CNBr-activated Sepharose 4 Fast Flow beads (GE Healthcare) according to the manufacturer’s instructions. For each column, 2 mg of GST or GST-HIF-1α531-826 was coupled to CNBr beads and incubated at 4°C overnight with 15 mg of protein lysate from NRCMs. Columns were subsequently washed and protein was eluted with 8M urea, dialyzed, and concentrated to > 3 mg/mL using a 10-kDa-exclusion Vivaspin column (Sartorius). The concentrated protein samples were fractionated by SDS-PAGE, stained for total protein using G250 Coomassie Brilliant Blue (BioRad) and gel bands were excised for analysis by MS.
Sepharose 4 fast flow beads
Manufactured by GE Healthcare
Sourced in United States
Sepharose 4 Fast Flow beads are a type of agarose-based chromatography media used for the purification and separation of biomolecules. They are characterized by their ability to facilitate rapid flow rates while maintaining high resolution during the chromatographic process.
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Lab products found in correlation
Vivaspin column, by Sartorius (1 mentions)
Protein g column, by GE Healthcare (1 mentions)
Protein g sepharose, by GE Healthcare (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Protino ni ted columns, by Macherey-Nagel (1 mentions)
Ni sepharose beads, by GE Healthcare (1 mentions)
Sds page, by GE Healthcare (1 mentions)
Modified porcine trypsin, by Promega (1 mentions)
16 protocols using sepharose 4 fast flow beads
1
HIF-1α Interacting Protein Capture
2
Quantifying BoNT/Hi Binding Kinetics
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For mAbs CR2, RAZ1 and 6F5.4, the solution phase affinity at equilibrium and binding kinetics were measured using flow fluorimetry in a KinExA (Sapidyne, Boise, ID, USA) as previously described [10 (link),25 (link)] except that BoNT/Hi toxin was used. BoNT/Hi toxin solution was studied at a concentration estimated to be >10-fold above the value of the equilibrium dissociation binding constant [KD] of the mAb for the toxin to generate a concentration-controlled curve for greater accuracy in measuring BoNT/Hi concentrations. mAb-containing solutions were serially diluted in a constant concentration of culture filtrate, from >10-fold above to <0.1-fold below the estimated BoNT/Hi concentrations, to capture a complete binding curve. After equilibrium was achieved, samples were passed over a flow cell packed with Sepharose 4 Fast Flow beads (GE Healthcare, Marlborough, MA, USA) covalently coupled with the measuring mAb. An Alexa-647-labeled mAb binding a nonoverlapping BoNT epitope was then passed over the flow cell, producing a signal proportional to the free BoNT in each sample. An analysis curve yielding values for KD and the binding concentrations of the BoNTs was generated using KinExA Pro software (version 4.2.14, Sapidyne, Boise, ID, USA) and a 1:1 reverse-binding model.
3
Purification of Anti-Malaria Antibodies
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The serum samples used in this study were pooled from eight P. vivax-infected patients from the ROK with a high response to the particular antigen of interest, which was screened by immunoscreening with a protein microarray. The control comprised pooled serum samples from eight healthy individuals from a nonendemic area of the ROK. Total IgG antibodies were purified from the pooled serum samples by using protein G columns according to the manufacturer’s protocol (GE Healthcare Life Sciences). The isolated IgG antibodies were dialyzed against RPMI 1640 medium and concentrated using centrifugal devices (Merck Millipore, Darmstadt, Germany) with a 30-kDa cut-off value to a concentration of 10 to 20 mg/mL. PvRBP1a, Pv41, and PvRhopH2 recombinant proteins (2–3 mg each) were immobilized on cyanogen bromide (CNBr)-activated Sepharose 4 fast flow beads (GE Healthcare Life Sciences) according to the manufacturer’s instructions. The total isolated IgG antibodies (0.5 mL) was loaded to the column filled with antigen coupled beads and eluted using an elution buffer (0.1 M glycine, pH 2.7). The eluted antigen-specific IgGs were immediately neutralized with a Tris buffer (pH 9.0) and dialyzed against incomplete RPMI 1640 medium to the desired concentration.
4
Crizotinib Affinity Probe Preparation
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The crizotinib affinity probe was prepared in two steps from commercially available 3-[(1RS)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyridin-2-amine (Selleckchem) according to literature and drug-affinity matrices were prepared as described previously (Huber et al., 2014). Briefly, affinity chromatography and elution were performed in duplicate using 25nmol of compound immobilized on 50μL NHS-activated Sepharose 4 Fast Flow beads (GE Healthcare Bio-Sciences) and 10mg total cell lysate as protein input per replicate. For competition experiments, cell lysates were pretreated with 20μM 3-[(1RS)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-[1-(piperidin-4-yl)-1H-pyrazol-4-yl]pyridin-2-amine for 30 minutes. Eluates were labeled with iTRAQ and quantitative protein mass spectrometry and bioinformatics analysis utilizing the R isobar package were performed as previously reported 48 (link),52 (link).
5
Affinity-based Protein Immobilization on NHS-Sepharose
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NHS-activated Sepharose 4 Fast Flow beads (GE Healthcare, 8 ml of slurry) were washed with 40 ml of 0.01 M HCl. Fifty milligrams of purified KKK_nl_gc_R7 in PBS (SI Fig. 1 ) were added and incubated for 2 h at RT. Absorbance of the flow-through at 280 nm was measured and showed that all protein had been coupled to the beads. Beads were washed with PBS and stored in PBS with 1 mM sodium azide at 4 °C where they were stable for several months.
6
Quantifying TEX101 Protein Variants
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Total TEX101 protein was enriched from SP and spermatozoa using an in-house anti-TEX101 mouse monoclonal antibody 34ED556. Briefly, protein G purified 34ED556 monoclonal antibody was immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose 4 Fast Flow beads (GE Healthcare). Fifty μl of beads (∼25 μg of 34ED556) in 0.1% BSA were incubated overnight at 4 °C with seminal plasma or spermatozoa lysate. After binding, beads were washed three times with tris buffer saline (50 mm Tris, 150 mm NaCl, pH 7.5) followed by washing with 50 mm ammonium bicarbonate. Proteins were digested overnight on beads using Glu-C. Supernatants were acidified with 1% TFA. Heavy peptides (200 fmol of WT and 500 fmol of G99V) were spiked into each sample after digestion. Digests were desalted, and peptides were measured by PRM assay. Raw files were analyzed with Skyline software (v3.6.0.10493), and the relative abundances of WT or G99V variant TEX101 forms were calculated using the light-to-heavy peptide ratios.
7
Co-IP and GST Pulldown Assays for Protein Interactions
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For co-IP assays, transfected cells were treated or not with 20 µM nocodazole (Sigma-Aldrich) or DMSO for 12 min and lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM EGTA, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with a cocktail of protease inhibitors (Roche). Co-IPs were performed with 600 µg precleared protein extracts, 2 µg GFP antibody (ab6556-25; Abcam), and 20 µl protein G Sepharose (GE Healthcare). GST-FIGNL1 and His-Fignl1Δ1–113-His were induced in BL21 Escherichia coli and purified on glutathione Sepharose 4 Fast Flow beads (GE Healthcare) or Protino Ni-TED columns (MACHEREY-NAGEL) according to the manufacturer’s instructions. For GST pulldown assays, COS-7 cells overexpressing EB3-GFP or GFP alone were lysed in RIPA buffer. Protein lysates or purified His-EB1-GFP were incubated with glutathione Sepharose 4 Fast Flow beads previously bound to purified GST-FIGNL1 or GST alone for 4 h at 4°C. Beads were washed several times in RIPA buffer and resuspended in 2× Laemmli buffer. Proteins retained on the beads were analyzed by WB by using GFP antibody.
8
Polyclonal Antibody Development for Meiotic Proteins
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Antibodies were raised against PRR19 C-terminal fragment (183 amino acids between Thr183 and Tyr366 residues) and CNTD1 C-terminal fragment (124 amino acids between Cys126 and Thr249 residues). Coding sequences corresponding to these peptides were cloned into pDEST17 bacterial expression vector. Recombinant 6xHis-tagged proteins were expressed in E. coli strain BL21 tRNA and subsequently purified by Ni-Sepharose beads and SDS-PAGE (Amersham, GE Healthcare). Either elution fractions or homogenised SDS-PAGE gel fragments containing the purified proteins were used for immunisation of rabbits and guinea pigs. PRR19 or CNTD1 fragments coupled to NHS-activated Sepharose 4 Fast Flow beads (Amersham, GE Healthcare) were used to affinity purify polyclonal antibodies following standard procedures. Goat anti-RNF212 was raised against full-length mouse RNF212 protein. The specificity of anti-PRR19, anti-CNTD1 and anti-RNF212 antibodies was confirmed by: (1) immunoprecipitations and western blot analysis of protein extracts from testes of wild-type and Prr19−/−, Cntd1−/− and Rnf212−/− mice, respectively; (2) immunostaining of spermatocyte nuclear surface spreads from testes of wild-type and mutant mice.
9
Affinity Purification of RNA-Protein Complexes
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N-hydroxysuccinimide-activated Sepharose 4 Fast Flow beads (GE Healthcare Life Science, Marlborough, MA, USA) were derivatized with 40 mM tobramycin following the manufacturer’s protocol. For affinity purification, the 4× binding buffer (80 mM Tris, pH 7.4/4 mM CaCl2/4 mM MgCl2/2 mM DTT) was freshly prepared. The tobramycin-coated sepharose beads were blocked with 500 μL of blocking buffer (1×BP/300 mM KCl/0.1 mg mL−1 tRNA/ 0.5 mg mL−1 BSA/ 0.01% Triton X-100) at 4 °C overnight. The beads were collected and incubated with cell lysates from 2 × 107 cells at 4 °C for 4 h. The beads were then isolated by centrifugation and washed with 1 mL of washing buffer (40 mM Tris/120 mM NaCl/1% TritonX-100, pH = 7.4) at room temperature for ten times. The bound RNA and proteins were eluted with 30 mM tobramycin solution. The HepG2-J6f1 cells were processed in parallel as the negative control. The proteins purified from HepG2-HULC-J6f1 cells were mixed with the control sample, separated on a 10% SDS-PAGE, and visualized using silver staining. Each gel lane was diced into ten slices, and in-gel tryptic digestion was conducted. Three biological replicates were analyzed for quantification.
10
FLNA Repeats Coupling for Affinity Purification
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Using pFASTBAC-FLNA vector (Nakamura et al., 2002 (link)) as the template, FLNA repeats 21-22 (test) and 1-2 (negative control) were cloned into pGEX4T-HT vector by PCR. The vectors were transformed into E. coli C41 (NEB) and protein expression was induced by 0.4 mM IPTG for 2 h. The proteins were affinity purified using glutathione beads and the GST-His tag was cleaved off by tobacco etch virus (TEV) protease. Purified FLNA repeats 21-22 and 1-2 were covalently coupled to NHS-activated Sepharose 4 Fast Flow beads (GE Healthcare) at 10 mg per 1 mL of the beads in PBS for 2 h at room temperature. The non-reacted groups of the beads were blocked with 0.1 M Tris-HCl pH 8.0 for 2 h at room temperature, equilibrated with TTBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% TritonX-100, 1 mM EGTA, 1 mM β-mercaptoethanol), and stored at 4°C.
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