ChIP experiments were performed as described previously with hypochlorite-isolated embryos or young adults (Guang et al. 2010 (link)). Animals were cross-linked in 2% formaldehyde for 30 min. Fixation was quenched with 0.125 M glycine for 5 min at room temperature. After cross-linking, samples were resuspended in FA buffer (50 mM Tris/HCl at pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl) with a proteinase inhibitor tablet (Roche, 04693116001) and sonicated for 20 cycles at medium output (each cycle: 30 sec on and 30 sec off) with a Bioruptor 200. Lysates were precleared and then immunoprecipitated with 1.5 µL of anti-GFP antibody (Abcam, ab290) for SNPC-4, TOFU-4, and TOFU-5 and 5 µL of anti-PRDE-1 for PRDE-1 overnight at 4°C. Antibody-bound complexes were recovered with Dynabeads Protein A. Following extensive sequential washes with 150, 500, and 1 M NaCl, DNA was treated with RNase (Roche) and ProK (New England Biolabs). Finally, resulting DNA samples were purified with QIAquick PCR purification kit (Qiagen, 28104).
Proteinase inhibitor tablet
Manufactured by Roche
Proteinase inhibitor tablet is a laboratory reagent used to inhibit the activity of proteolytic enzymes, known as proteases, in biological samples. It is designed to prevent the degradation of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Anti gfp antibody, by Abcam (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Irdye 800 goat antirabbit, by Rockland Immunochemicals (1 mentions)
Goat anti rabbit irdye 680, by LI COR (1 mentions)
β actin, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Gfp trap a, by Proteintech (1 mentions)
Ab9045, by Abcam (1 mentions)
Pd l1 e1l3n, by Cell Signaling Technology (1 mentions)
Thermomixer, by Eppendorf (1 mentions)
5 protocols using proteinase inhibitor tablet
1
ChIP Experiments for C. elegans Proteins
2
Histone Extraction and Western Blot
3
GFP-Trap Protein Isolation from Nicotiana
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Proteins were isolated from N. benthamiana leaves 2 days after agroinfiltration. Leaf material was grinded in liquid nitrogen, of which one gram of grinded sample was incubated with 2 mL of extraction buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1 % NP-40, 5 mM DTT, and 1 proteinase inhibitor tablet (Roche) per 50 mL) for 30 min on ice. Cell debris was pelleted and 100 μl supernatant was kept as input sample. Remaining volumes were incubated with 15 μl GFP-trap_A (ChromoTek) beads at 4 °C for 2 h. Subsequently, the beads were collected by centrifugation at 720 g for 2 min and washed 8 times with extraction buffer. After washing, the beads were collected and pipetted into a new 1.5 mL tube containing 100 μl extraction buffer. Protein samples were boiled in 4х loading buffer (200 mM Tris-HCl pH 6.8, 8 % SDS, 400 mM DTT, 40 % glycerol and 0.2 % bromophenol blue) for 5 min before gel electrophoresis.
4
Chromatin Immunoprecipitation (ChIP) in C. elegans
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ChIP experiments were performed as previously described with L4 staged animals. After crosslinking, samples were resuspended in 1 ml FA buffer (50 mM Tris/HCl [pH 7.5], 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, and 150 mM NaCl) with a proteinase inhibitor tablet (Roche no. 05056489001) and sonicated for 20 cycles at high output (each cycle: 30 s on and 30 s off) with a Bioruptor plus. Lysates were precleared and then immunoprecipitated with 2 μl anti-histone H3 (monomethyl K9) antibody (Abcam no. ab9045), 2 μl anti-histone H3 (dimethyl K9) antibody (Abcam no. mAbcam1220) or 2 μl anti-trimethylated H3K9 antibody (Millipore no. 07-523). ChIP signals were normalized to coimmunoprecipitated ama-1 and then expressed as fold changes relative to that of daf-2 (e1370) animals. qRT-PCR primers for ChIP assays are listed in Supplementary file 5 .
5
Monocyte Lysis and Protein Analysis
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Cells were harvested, washed once with PBS, and then lysed. Lysis was mediated through two separate methods depending on cell number harvested. For samples less than or equal to 5 × 106 monocytes, cells were resuspended in 40 microliters of a solution of 50 ml NP-40 buffer (0.1% NP-40, 150 mM NaCl, 50 mM Tris-HCl [pH 8.0] containing a proteinase inhibitor tablet [Roche], 30 mM β-glycerophosphate, 50 mM NaF, and 1 mM Na3VO4) and subsequently frozen and thawed once to complete lysis. For samples more than 5 × 106 monocytes, cells were resuspended in 2× Laemmli sample buffer and simultaneously boiled and vortexed at 1,200 rpm in a ThermoMixer (Eppendorf) until the pellet was no longer visible (10 to 20 min). Equal amounts of protein were loaded per lane, resolved by SDS-PAGE, and then transferred to a nitrocellulose membrane. The antibodies used were PD-L1 (E1L3N; Cell Signaling Technology), ORF45 (2D4A5; Thermo Fisher), and actin (C-11; Santa Cruz).
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