Proteins from 5-week-old SnRK1α1–GFP, SnRK1α1ΔKA1–GFP, SnRK1α1–GFPsiz1-2 or 35S::GFP plant leaves were extracted with immunoprecipitation (IP) buffer [50 mM Tris–HCl pH 8.0, 50 mm NaCl, 1% (V/V) Igepal CA-630, 0.5% (w/V) sodium deoxycholate, 0.1% (w/V) SDS, 1 mM EDTA pH 8.0, 50 μM MG132, 50 mM N-ethylmaleimide and cOmplete protease inhibitor cocktail (one tablet/10 mL)]. After clearing samples by centrifugation (6785 g , 2°C, 10 min) 800 μL of supernatant were supplemented with fresh MG132 (50 μm ) and incubated at 4°C for 1 h with 40 μL of μMACS anti–GFP MicroBeads (μMACS GFP Isolation Kit, Miltenyi, 130-091-125). Samples were thereafter loaded in μColumns (Miltenyi Biotec, Bergisch Gladbach, Germany 130-042-701) pre-equilibrated with 1 mL of IP buffer, and allowed to flow through. Columns were washed three times with 200 μL and once with 600 μL of IP buffer and proteins eluted with 80 μL of elution buffer (Miltenyi, 130-091-125) at 95°C. β-Mercaptoethanol (2%) was added to the eluates prior to boiling for 5 min at 95°C. Proteins were resolved by SDS-PAGE, wet-transferred to a PVDF membrane (30 V, 16 h at 4°C), and analysed by immunoblotting with SnRK1α1, SnRK1β1, SnRK1γ, SnRK1βγ, SUMO1, UBQ11 and GFP antibodies. For each GFP immunoprecipitation experiment, immunodetection with different antibodies was done using equal loading on independent membranes.
Elution buffer
Manufactured by Miltenyi Biotec
Elution buffer is a laboratory solution used to extract and recover target molecules, such as proteins or nucleic acids, from a solid support or affinity matrix during the purification process. It facilitates the release and collection of the desired analyte for further analysis or downstream applications.
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3 protocols using elution buffer
1
SnRK1 Protein Interactome Profiling
2
Nuclear Proteome Isolation and Analysis
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Plants were grown in 12-h light/12-h dark cycles and harvested on Day 10 at ZT3 or ZT17. Approximately 7.5 g of vegetative tissue was flash frozen in liquid nitrogen and ground into fine powder with a mortar and pestle. Ground tissue was resuspended in nuclei enrichment buffer [50 mM Tris pH 7.5, 400 mM sucrose, 2.5 mM MgCl 2 , 1 mM DTT, 1 mM PMSF, cOmplete Protease Inhibitor cocktail (Roche)], the nuclear pellet was collected, resuspended in IP buffer [100 mM Tris pH 7.5, 1 mM EDTA, 75 mM NaCl, 10% glycerol, 0.3% Triton X-100, 0.05% SDS, 10 mM MG-132, 1 mM PMSF, cOmplete Protease Inhibitor cocktail (Roche)] and pelleted again. Nuclei were resuspended in IP buffer, disrupted via sonication, and adjusted to 1 mg mL -1 in IP buffer. One milligram of extract was incubated with µMACS MicroBeads conjugated to an anti-HA monoclonal antibody (Miltenyi Biotec) and beads were then captured on µMACS M-columns. Beads were washed 3× with ice cold IP buffer and 1× with ice cold TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA). Proteins were eluted off the beads with elution buffer (Miltenyi Biotec). Proteins were resolved using SDS polyacrylamide gel electrophoresis and gels were sent to the Rutgers Biological Mass Spectrometry Facility, Robert Wood Johnson Medical School. Proteins were eluted and subjected to MS as previously described (Wei et al. 2020) .
3
Co-immunoprecipitation of Cyanobacterial Proteins
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For co-immunoprecipitations of fluorescently tagged CCRP candidates, cyanobacterial strains expressing YFP-All4981 or HmpFSyn-YFP were grown in BG11 or BG110 liquid medium. About 20–30 ml of the respective culture was pelleted by centrifugation (4800 × g, 10 min, RT), cells were washed twice by centrifugation (4800 × g, 10 min, RT) with 40 ml PBS and then resuspended in 1 ml lysis buffer (PBS-N: PBS supplemented with 1% NP-40) supplemented with protease inhibitor cocktail (PIC; cOmplete™, EDTA-free Protease Inhibitor Cocktail, Sigma-Aldrich). Cells were lysed using the VK05 lysis kit (Bertin) in a Precellys® 24 homogenizer (3 strokes for 30 seconds at 6500 rpm) and cell debris was pelleted by centrifugation (30 min, 21,100 × g, 4 °C). 50 µl μMACS anti-GFP MicroBeads (Miltenyi Biotec) was added to the resulting cell-free supernatant and incubated for 1 h at 4 °C with mild rotation. Afterwards, the sample was loaded onto µColumns (Miltenyl Biotec), washed two times with 1 ml lysis buffer and eluted in 50 µl elution Buffer (50 mM Tris HCl pH 6.8, 50 mM DTT, 1% SDS, 1 mM EDTA, 0.005% bromphenol blue, 10% glycerol; Miltenyl Biotec). Until further use, samples were stored at −80 °C. Proteins were identified by mass spectrometry. A detailed protocol of the mass spectrometry analysis is available upon request from the authors.
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