Parasitized erythrocytes were transferred to RPMI1640 medium supplemented with 25% fetal bovine serum, 0.05 mg/mL penicillin and 0.05 mg/mL streptomycin. The parasitized erythrocytes were incubated for 22 h in 90% N2, 5% CO2 and 5% O2. Mature schizonts and gametocytes were harvested by Nycodenz density gradient centrifugation, as described previously [7 (link)]. Proteins were extracted using Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. Protein IP in transgenic parasites expressing mCherry fused to PBANKA_1019700 was performed using RFP-Trap Agarose and a RFP-Trap-A kit, according to the manufacturer’s instructions (Chromotek, Planegg, Germany).
Rfp trap agarose
Manufactured by Proteintech
Sourced in Germany
RFP-Trap Agarose is a pre-packed agarose resin designed for the affinity purification of RFP-tagged proteins from cell lysates. The agarose beads are covalently coupled with anti-RFP nanobodies, providing a robust and specific method for the capture and isolation of RFP-fusion proteins.
Automatically generated - may contain errors
Lab products found in correlation
Mammalian protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Gfp trap agarose, by Proteintech (2 mentions)
Gfp trap a kit, by Proteintech (1 mentions)
Timstof pro mass spectrometer, by Bruker (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Immobilon pvdf membrane, by Merck Group (1 mentions)
Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti mouse hrp conjugated secondary antibody, by Vector Laboratories (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
8 protocols using rfp trap agarose
1
Purification of Malaria Parasite Proteins
2
Protein Extraction and IP from Malaria Parasites
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Infected erythrocytes were transferred to RPMI1640 medium supplemented with 25% fetal bovine serum, 0.05 mg/mL penicillin and 0.05 mg/mL streptomycin. The infected erythrocytes were incubated for 22 h in 90% N2, 5% CO2 and 5% O2. Mature schizonts and gametocytes were harvested by Nycodenz density gradient centrifugation, as described previously (Niikura et al., 2020 (link)). Proteins were extracted using Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. Protein IP in transgenic parasites expressing mCherry fused to GBP2 or NAB2 was performed using GFP- or RFP-Trap Agarose and a GFP-Trap-A kit, according to the manufacturer’s instructions (Chromotek, Planegg, Germany).
3
Affinity Purification and Quantitative Proteomics
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Leaves were homogenized, centrifuged, and mixed with 25 µL RFP-Trap Agarose or GFP-Trap Agarose (ChromoTek). Beads were spun down, washed, and stored at −80 until mass spectrometry analysis. The washed beads were incubated for 30 min with elution buffer. Tryptic peptide mixtures were loaded on Evotips (Evosep) and peptides were separated and injected via a CaptiveSpray source and 10-μm emitter into a timsTOF pro mass spectrometer (Bruker) ran in PASEF mode (89 (link)).
Raw mass spectrometry data were analyzed with MaxQuant against the Arabidopsis Uniprot FASTA database. Statistical analyses of LFQ-derived protein expression data were performed using the automated analysis pipeline of the Clinical Knowledge Graph (90 (link)). Differentially expressed proteins in each group comparison were identified by SAMR multiclass test with permutation-based FDR correction for multiple hypothesis, followed by post hoc pairwise comparison unpaired t tests using the same parameters and permutation-based FDR correction (91 ). Significantly regulated proteins are colored in red and blue in the volcano plots for up and down-regulated proteins, respectively.
Raw mass spectrometry data were analyzed with MaxQuant against the Arabidopsis Uniprot FASTA database. Statistical analyses of LFQ-derived protein expression data were performed using the automated analysis pipeline of the Clinical Knowledge Graph (90 (link)). Differentially expressed proteins in each group comparison were identified by SAMR multiclass test with permutation-based FDR correction for multiple hypothesis, followed by post hoc pairwise comparison unpaired t tests using the same parameters and permutation-based FDR correction (91 ). Significantly regulated proteins are colored in red and blue in the volcano plots for up and down-regulated proteins, respectively.
4
Immunoblotting Protocol for RFP-Trap Pulldown
5
Viral Protein-Host Interaction Mapping
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Co‐IP assays were performed as described in the protocol for RFP‐Trap® Agarose (Chromotek, Martinsried, Germany). Briefly, RFP‐p33, which acts as a bait, was co‐expressed with free mGFP, CmMLP2‐mGFP, or CmM∆Cys‐mGFP by agroinfiltration, respectively, in N. benthamiana. Agrobacterium culture harbouring wild‐type CTV (OD600 = 0.1) construct was introduced simultaneously to create a similar cellular environment with the physiological viral infection. At three dpi, the total protein was extracted from N. benthamiana leaves and used to confirm the expression of the respective proteins as the input control. The RFP‐Trap® Agarose beads were applied to capture the RFP‐p33 protein. The eluted protein complexes were collected as output. The extracted proteins (input and output) were further separated on a 10% SDS‐polyacrylamide gel and detected by immunoblot using anti‐p33 (Ab:p33) or anti‐GFP antibodies (Ab:GFP, Abcam, Cambridge, UK).
6
Immunoprecipitation of Huntingtin Protein
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HEK293T cells were grown in T75 flasks 2 × 106 cells/flask and transfected with either 15 µg of flKMO-RFP or RFP using Lipofectamine 3000 (Thermo Fisher) for 48 h. Cells were washed twice with ice-cold PBS and lysed for 30 min in 1 mL of ice-cold lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.05 % n-dodecyl β-D-maltoside, 1% (v/v) HALT protease inhibitor cocktail, 1% (v/v) HALT phosphatase inhibitor cocktail (Thermo Fisher Scientific, Loughborough, UK). Four aliquots of each sample (250 µL) were diluted 1:1 with dilution buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1% (v/v) HALT protease inhibitor cocktail, 1% (v/v) HALT phosphatase inhibitor cocktail) and lysates were cleared with Binding Control Agarose (20 µL per aliquot) (Chromotek, Manchester, UK) for 30 min at 4 °C with rotation. The supernatant was then incubated with 25 µL of RFP-Trap Agarose (Chromotek, Manchester, UK) for 1 h at 4 °C with rotation. The agarose beads were washed thrice with 500 µL wash buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA). Proteins were eluted in 30 µL 2× SDS-PAGE loading buffer and were separated by 10% SDS-PAGE and then transferred onto nitrocellulose membranes. The membranes were incubated with HTT antibody followed by an anti-mouse HRP conjugated secondary antibody (1:5000; Vector Laboratories, Peterborough, UK).
7
Recombinant Protein Expression in N. benthamiana and A. thaliana
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For recombinant expression of identified proteins in N. benthamiana, syringe-mediated leaf infiltration of 5-week-old plants was used. Agrobacterium tumefaciens strain UIA143 (Strasser et al., 2005 (link)) carrying the respective plasmid for expression was grown over night at 29°C and adjusted to an OD600 of 0.15 on the next day in infiltration buffer (28 mM glucose, 50 mM 2-(4-morpholino)-ethanesulphonic acid (MES), 2 mM Na3PO4.12H2O, 0.1 mM acetosyringone). p29-Fc-KOR1 was additionally co-infiltrated with silencing suppressor p19 (Garabagi et al., 2012 (link)) at an OD600 of 0.1. Leaf material was collected 2 days post infiltration (dpi) and snap-frozen. For stable expression in A. thaliana, plants were transformed using the floral dip method (Clough and Bent, 1998 (link)). Stems of homozygous plants were collected ~5 weeks after sowing. Plant material was disrupted mechanically, and total proteins extracted using RIPA buffer. Recombinantly produced glycoproteins were purified via their fused tags, either GFP for KORRIGAN (GFP-Trap agarose, Chromotek) or mRFP for the other constructs (RFP-Trap agarose, Chromotek). After capture, proteins were eluted in 1.5x Laemmli buffer.
8
Immunoprecipitation and SUMO Immunoblotting
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Cell lysate was prepared by bead beating as described for immunoprecipitation above, and TAP-tagged or mCherry-tagged proteins were immunoprecipitated using IgG Sepharose or RFP-Trap agarose (Chromotek). Then immunoprecipitated proteins were washed and eluted with SDS loading buffer before separating by NuPAGE gel (for Top2, 6% separation gel) and immunoblotting with anti-SUMO, and PAP or anti-mCherry antibodies.
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