Oligonucleotides and expression plasmids used for cloning are listed in Supplementary Tables 7 and8, respectively. For plant transformation, the URT1 sequence was PCR-amplified from genomic DNA and includes 5' UTR (URT1-myc constructs) or 3' UTR (myc-URT1 and YFP-URT1 constructs). For bacterial expression, URT1, DCP5 and CAF1b sequences were PCR-amplified from cDNA templates. 2e. For IPs without crosslinking on Arabidopsis samples, 300 mg of flower buds or seedlings were ground in 1.5 ml of ice-cold lysis buffer (50 mM Tris-HCl pH 8, 50 mM NaCl, 1 % Triton X-100, protease inhibitors (cOmplete, EDTA-free Protease Inhibitor Cocktail, Roche)). After cell debris removal by centrifugation (twice 10 min at 16,000 g, 4°C) supernatants were incubated for 30 min with 50 µl of magnetic microbeads coupled to anti-c-myc antibodies or anti-GFP antibodies (Miltenyi). Beads were loaded on magnetized µMACS separation columns equilibrated with lysis buffer and washed four times with 200 µl of washing buffer (20 mM Tris-HCl pH 7.5, 0.1 % Triton X-100). Samples were eluted in 100 µl of pre-warmed elution buffer (50 mM Tris-HCl pH 6.8, 50 mM DTT, 1 % SDS, 1 mM EDTA, 0.005 % bromophenol blue, 10 % glycerol). Negative control IPs were performed under the exact same conditions with Col-0 plants.
Anti gfp antibody
Manufactured by Miltenyi Biotec
Sourced in Germany
The Anti-GFP antibody is a laboratory reagent designed to detect and bind to the green fluorescent protein (GFP) molecule. It can be used in various applications that involve the identification or quantification of GFP-tagged proteins or cells.
Automatically generated - may contain errors
Lab products found in correlation
Edta free protease inhibitor cocktail, by Roche (1 mentions)
Anti p44 42 mapk antibody, by Cell Signaling Technology (1 mentions)
Anti actin antibody, by ABclonal (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Anti mcherry antibody ab167453, by Abcam (1 mentions)
Paramagnetic beads, by Miltenyi Biotec (1 mentions)
Complete protease inhibitors 1x, by Roche (1 mentions)
7 protocols using anti gfp antibody
1
Immunoprecipitation of Arabidopsis Proteins
2
Arabidopsis PSY-GFP Protein Purification
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CoIP was conducted with quadruplicate biological replicates as described previously (Zhou et al., 2015) . In brief, proteins were extracted from Arabidopsis plants expressing 35S:PSY-GFP or 35S: GFP, mixed with magnetic beads conjugated to anti-GFP antibodies (Miltenyi Biotec, Auburn, CA), and incubated on ice for 30 min. Protein complexes containing PSY-GFP and GFP were purified in m columns by washing four times with extraction buffer and eluting with 23 SDS loading buffer.
3
Protein Extraction and MAPK Phosphorylation Analysis
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Total proteins were extracted using extraction buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2% Triton X-100, and 1× Protease Inhibitor Cocktail. To detect MAPK phosphorylation, 1 nM calyculin A and 25 mM NaF were added to the extraction buffer. The green fluorescent protein (GFP) was detected using an anti-GFP antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), and phosphorylation of MAPK was detected using an anti-p44/42 MAPK antibody (Cell Signaling Technology, Danvers, MA, USA). Anti-actin antibody (ABclonal Biotechnology, Wuhan, China) served as a loading control [62 ].
4
Immunoblot analysis of plant proteins
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Arabidopsis seedlings or infiltrated tobacco leaves were snap-frozen in liquid nitrogen and homogenized with mortar and pestle. The plant extract was resuspended in 50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% (vol/vol) Triton X-100, and cOmplete TM EDTA-free Protease Inhibitor Cocktail (Merck). 50 μg of protein extract (estimated by Bradford, Bio-Rad), pre-boiled for 5 min, was run on a 10% SDS–PAGE gel. Blotting was performed on a nitrocellulose membrane (GE Healthcare). After blocking with TBS buffer supplemented with 0.1% (vol/vol) Tween 20 and 5% (wt/vol) powder milk, the membrane was first incubated for 1 h with an anti-mCherry antibody (ab167453, dilution 1:2,000; Abcam), and then with an anti-rabbit peroxidase conjugate antibody (dilution 1:10,000, 1 h; Calbiochem). For mCitrine detection, the membrane was incubated for 1 h with an anti-GFP antibody coupled with HRP (Miltenyi Biotec) at 1:2,000 dilution. RuBisCO proteins were visualized with Ponceau (0.1% [wt/vol] Ponceau S in 5% [vol/vol] acetic acid) as loading controls.
5
Chitin-binding Protein Extraction and Detection
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Total proteins were extracted from N. benthamiana leaves. Chitin-binding proteins were pulled down using chitin magic beads according to the manufacturer’s protocol (New England Biolabs, Ipswich, MA, USA) and then detected with an immunoblot assay using an anti-GFP antibody (Miltenyi Biotec).
6
UV-B Induced UVR8 Dimer-Monomer Dynamics
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Agrobacterium cells transformed with the appropriate plasmid were infiltrated into the lower side of N. benthamiana leaves using a syringe, as described by Heilmann et al. (2016) . The infiltrated plants were placed in a growth room at 28°C and left for 2-3 d before treatment. Plants were transferred to darkness for 16 h and then exposed to either 20 lmol m À2 s À1 white light or 3 lmol m À2 s À1 narrowband UV-B for 3 h. UVR8 dimer/ monomer status was examined as described above following protein extraction according to Heilmann et al. (2016) and incubation of western blots with an anti-GFP antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) . The cellular localisation of GFP fluorescence was examined using a Leica TCS SP8 (SMD FSU) confocal microscope.
7
Co-Immunoprecipitation of RGA and IDD2 in N. benthamiana
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CoIP assays were performed on N. benthamiana agro-infiltrated leaves with p35S::IDD2-RFP, p35S::RGA-GFP or p35S::RGA m2 -GFP. Three days after infiltration, total proteins were extracted with the native extraction buffer [Tris-HCl (pH 7.5) 50 mM, glycerol 10%, Nonidet P-40 0.1% supplemented with Complete Protease Inhibitors 1X (Roche)], and then incubated for 2h at 4°C with 50 µl of anti-GFP antibody conjugated with paramagnetic beads (Miltenyi Biotec). After incubation, samples were loaded onto a magnetic column system (µ columns ;
Miltenyi Biotec) to recover the immunoprotein complexes according to manufacturer's protocol. The immunoprecipitated (RGA-GFP and RGA m2 -GFP) and co-immunoprecipitated (IDD2-RFP) proteins were detected by western-blot with anti-GFP (JL8; Clontech) and anti-RFP (6G6; Chromotek), respectively.
Miltenyi Biotec) to recover the immunoprotein complexes according to manufacturer's protocol. The immunoprecipitated (RGA-GFP and RGA m2 -GFP) and co-immunoprecipitated (IDD2-RFP) proteins were detected by western-blot with anti-GFP (JL8; Clontech) and anti-RFP (6G6; Chromotek), respectively.
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