Mixed-stage worms containing either Pvgln-1::vgln-1::gfp (kuIs102 ) (strain MH4920) or Pvgln-1::gfp (kuIs103 ) (strain MH4922) were grown on NGM plates with HB101 bacteria, collected by washing with M9 buffer, and irradiated by UV (Broughton and Pasquinelli 2013 (link)) to crosslink protein to RNA, and then stored at −70°. Worms were lysed by grinding in liquid nitrogen and immediately resuspended in lysis buffer (10 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, plus addition of complete mini Roche protease inhibitor pills, Thermo Fisher RNaseOut, 100 mM PMSF). Immunoprecipitation of protein crosslinked to RNA was performed with GFP-Trap A fusion proteins coupled to agarose beads according to the vendor’s protocol (Chromotek). RNA was digested, radiolabeled, and visualized as described (Moore et al. 2014 (link)).
Agarose beads
Manufactured by Proteintech
Sourced in Germany
Agarose beads are a type of chromatography resin used for the purification and separation of biological molecules, such as proteins, nucleic acids, and other biomolecules. They consist of cross-linked agarose polymer chains that form a porous matrix, allowing for the efficient capture and elution of the target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Protein a g agarose bead, by Beyotime (3 mentions)
Mops buffer, by Thermo Fisher Scientific (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Gfp trap, by Proteintech (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Protein a sepharose beads, by GE Healthcare (1 mentions)
Gfp trap agarose beads, by Proteintech (1 mentions)
Anti gfp, by Novus Biologicals (1 mentions)
Glutathione sepharose, by GE Healthcare (1 mentions)
Gfp trap a beads, by Proteintech (1 mentions)
Mg132, by Merck Group (1 mentions)
Protein g sepharose, by GE Healthcare (1 mentions)
Branson sonifier 250, by Emerson (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ab290, by Abcam (1 mentions)
Ubiquitin, by Proteintech (1 mentions)
Ab45171, by Abcam (1 mentions)
Anti ubiquitin, by Proteintech (1 mentions)
10 protocols using agarose beads
1
Crosslinking and Immunoprecipitation of Worm RNA-Protein Complexes
2
GFP Immunoprecipitation and Western Blotting
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GFP immunoprecipitations were carried out overnight at 4 °C with GFP-Trap coupled to agarose beads (ChromoTek) in the presence of 150 mM NaCl and 0.5% Nonidet-P40 substitute. For the washes 300 mM NaCl was used.
For western blot analysis, extracts or immunoprecipitated proteins were run on NuPAGE bis–tris 4–12% gels in MOPS buffer (Invitrogen) and transferred to nitrocellulose membrane (Protran). Primary antibodies were detected using horse-radish-peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) and SuperSignal enhanced chemiluminescent detection kit (Pierce). The molecular weights indicated on the western blots correspond to the PageRuler Prestained Protein Ladder (26616, Thermo Scientific) except the 240 kDa which corresponds to CABIN1 molecular weight. Uncropped scans of the blots shown in the main figures are provided in supplementary Figs.9 , 10 and 11 .
For western blot analysis, extracts or immunoprecipitated proteins were run on NuPAGE bis–tris 4–12% gels in MOPS buffer (Invitrogen) and transferred to nitrocellulose membrane (Protran). Primary antibodies were detected using horse-radish-peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) and SuperSignal enhanced chemiluminescent detection kit (Pierce). The molecular weights indicated on the western blots correspond to the PageRuler Prestained Protein Ladder (26616, Thermo Scientific) except the 240 kDa which corresponds to CABIN1 molecular weight. Uncropped scans of the blots shown in the main figures are provided in supplementary Figs.
3
Immunoprecipitation and Western Blotting Protocol
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Cells were washed with ice-cold PBS and lysed with IP lysis buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 5 mM EDTA and 0.5% (v/v) Triton X-100, supplemented with ‘cOmplete’ protease inhibitors (Roche)). The lysates were centrifuged at 50,000 rpm for 30 min at 4°C. The supernatants were pre-cleared for 1 hr at 4°C with agarose beads (Chromotek), then incubated with GFP-Trap agarose beads (Chromotek) for 1 hr at 4°C. Proteins were eluted from the beads with sample buffer for 10 min at 95°C. Samples were subjected to western blotting.
For pulse chase samples, cells were washed with ice-cold PBS and lysed with IP lysis buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100 and 1% (w/v) octyl-glucoside, supplemented with protease inhibitors). The lysates were centrifuged at 20,000 g for 20 min at 4°C. The supernatants were pre-cleared for 1 hr at 4°C with Protein A Sepharose beads (GE Healthcare), then incubated with Protein A Sepharose beads and rabbit anti-caveolin1 antibody overnight at 4°C. Proteins were eluted from the beads with sample buffer for 10 min at 95°C.
For pulse chase samples, cells were washed with ice-cold PBS and lysed with IP lysis buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100 and 1% (w/v) octyl-glucoside, supplemented with protease inhibitors). The lysates were centrifuged at 20,000 g for 20 min at 4°C. The supernatants were pre-cleared for 1 hr at 4°C with Protein A Sepharose beads (GE Healthcare), then incubated with Protein A Sepharose beads and rabbit anti-caveolin1 antibody overnight at 4°C. Proteins were eluted from the beads with sample buffer for 10 min at 95°C.
4
GST-pulldown and GFP-trap Assays
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Protein G sepharose and glutathione sepharose were obtained from GE Healthcare (Freiburg, Germany). GFPtrapA beads comprise a single domain GFP antibody immobilized on agarose beads (Chromotek, Martinsried, Germany). Proteasome inhibitor MG132 was purchased from Sigma (Munich, Germany). The following antibodies were used: anti-tubulin, mouse IgG (Sigma); anti-GFP (Novus Biologicals, CO); anti-PML H-238, anti-SIAH-1 N-15 (Santa Cruz), anti-HSV1/2 VP5 H1.4 (Acris, Herford, Germany); anti-ICP0 R147 and anti-ICP27 R152 (Biomedizinische Forschungsgesellschaft mbH Vienna, Austria), and anti-GST-DX700 (IRDye® labeled antibodies; Rockland Immunochemicals, Gilbertsville, CA). For infrared fluorescence detection (Li-COR Biosciences, Bad Homburg, Germany) secondary antibodies coupled to IRDye® fluorophores (Rockland) were used. glutathione sepharose for GST-pulldown experiments was obtained from GE Healthcare (Freiburg, Germany).
5
GFP Pull-Down Assay with Alpaca Antibodies
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GFP pull down with agarose beads coupled to Alpaca α-GFP antibodies (Chromotek, Planegg, Germany) was done per manufacturer's instructions with some modifications. Briefly, transfected HEK293T cells transiently expressing SINC-GFP (1 × 150–mm dish) and mock-transfected cells were washed thrice with PBS and lysed in 200 μl of lysis buffer (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40). Cells were placed on ice for 30 min with extensive pipetting every 10 min and sonicated once (5–10 s) in a Branson Sonifier 250 (Branson Ultrasonics, Danbury, CT). Lysates were spun at 20,000 × g for 10 min, and the supernatants were diluted in wash buffer (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA) to 1 ml final volume before incubation with prewashed GFP-Trap-A for 2 h at 4°C. Beads were washed three times with ice-cold wash buffer, and immunocomplexes were disassociated by boiling in SDS–PAGE sample buffer before analysis by immunoblot.
6
Isolation and Analysis of MoHRD1-GFP Fusion Proteins
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The MoHRD1-GFP fusion construct was introduced by transformation
into protoplasts of the Mohrd1 null mutant. Mycelia were ground
into fine powder in liquid nitrogen and resuspended in 20 mL lysis buffer (10 mM
Tris-HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) with freshly added 1 mM
phenylmethylsulfonyl fluoride (PMSF) and 10 μL of protease inhibitor cocktail
(Sigma Aldrich , Shanghai, China). Then, total proteins were incubated with
anti-GFP (green fluorescent protein) agarose beads (ChromoTek,
Planegg-Martinsried, Germany). Proteins bound to GFP agarose beads were eluted
after a series of washing steps according to the manufacturer’s protocol.
Analysis of bound proteins was performed by the Beijing Genomics Institute using
MS.
into protoplasts of the Mohrd1 null mutant. Mycelia were ground
into fine powder in liquid nitrogen and resuspended in 20 mL lysis buffer (10 mM
Tris-HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) with freshly added 1 mM
phenylmethylsulfonyl fluoride (PMSF) and 10 μL of protease inhibitor cocktail
(Sigma Aldrich , Shanghai, China). Then, total proteins were incubated with
anti-GFP (green fluorescent protein) agarose beads (ChromoTek,
Planegg-Martinsried, Germany). Proteins bound to GFP agarose beads were eluted
after a series of washing steps according to the manufacturer’s protocol.
Analysis of bound proteins was performed by the Beijing Genomics Institute using
MS.
7
Immunoprecipitation of Fluorescent Fusion Proteins
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Immunoprecipitation of mCitrine and GFP fusion proteins was done using GFP-TrapA nanobody coupled to agarose beads (Chromotek, Germany) following the manufacturer’s protocol. Cells lysate were prepared in RIPA buffer and the second beads wash following the binding was done in higher salt stringency buffer (500 mM NaCl instead of 150 mM). One tenth of lysate volume of bound and unbound fractions were loaded onto 12% SDS-PAGE gels, blotted onto nitrocellulose membranes, and probed with mouse monoclonal anti-PMP70 (1:500), mouse monoclonal anti-ALDP (BD Bioscience 2D-ALD6, 1:500), polyclonal rabbit anti-PEX14 (1:500), or rabbit polyclonal anti-GFP (Abcam Ab290, 1:2000). HRP-conjugated monoclonal donkey anti-mouse antibody (1:5000) and polyclonal donkey anti-rabbit antibody (1:5000) were used to detect probed proteins. Protein bands were detected using LumiLight PLUS and scanned by LAS4000 analyzer.
8
Immunoprecipitation of Ubiquitin in CaOV3 Cells
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Control, LINC00452- overexpressing and knockdown CaOV3 cells from 10-cm culture dishes were lysed with RIPA buffer, respectively. Human IgG (Bioss, #bs-0297P, 1:150) or ROCK1 antibody (Abcam, #ab45171, 1:50) were incubated separately with each cell lysate overnight at 4°C with gentle rotation. 20 μl Protein A/G agarose beads (Beyotime Biotechnology, #P1012) were added and incubated at 4°C with gentle rotation for 2 h. After the sequential wash with PBS and cell lysis buffer, the agarose beads were resuspended in 20 μl 1x SDS-PAGE loading buffer, and boiled for western blot analysis on Ubiquitin (Proteintech, 10201-2-AP, 1:200).
9
Immunoprecipitation and Western Blot Analysis
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BLCA cells grown in 10-cm culture dishes were lysed with RIPA buffer. Human IgG (Bioss, #bs-0297P, 1:150), anti-FTO antibody (RD, #MAB7208, 1:50) or anti-PYCR1 antibody (Abcam, #ab226340, 1:100) were incubated separately with each cell lysate overnight at 4° C with gentle rotation. 20 μl Protein A/G agarose beads (Beyotime Biotechnology, #P1012) were added and incubated at 4° C with gentle rotation for 2 h. After the sequential wash with PBS and cell lysis buffer, the agarose beads were resuspended in 20 μl 1x SDS-PAGE loading buffer, and boiled for western blot analysis on Ubiquitin (Proteintech, 10201-2-AP, 1:200) or USP18 (Abcam, #ab168478, 1:500).
10
Protein Ubiquitination Profiling
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Transfected cells were lysed with RIPA buffer. IgG or HDAC1 antibody was respectively incubated with cell lysates overnight, accompanied by gentle rotation. Then protein A/G agarose beads (Beyotime Biotechnology, Shanghai, China) were added for incubation. After washing, the agarose beads were re-suspended in SDS-PAGE buffer, and boiled for western blot analysis using anti-ubiquitin (Proteintech, Rosemont, IL, USA).
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