The RIP assay was performed using an EZ-Magna RIP Kit (EMD Millipore) according to the manufacturer's instructions. NP cell lysates were lysed with RIPA buffer (Beyotime Institute of Biotechnology) for 5 min at 4°C. Antibodies including anti-Argonaute 2 (anti-Ago2; cat. no. ab186733; 1:50; Abcam) and anti-Immunoglobulin G (anti-IgG; cat. no. 12-370; 1:100; EMD Millipore) were incubated with protein A/G magnetic beads (Pierce; Thermo Fisher Scientific, Inc.) for 1 h at 4°C. Then, cell lysate was mixed with the beads to incubate for 4 h at 4°C. Beads were washed twice using PBS buffer (Sangon Biotech Co., Ltd.), and the mixture was centrifuged at 2,500 × g for 10 min at 4°C. RNA was purified with 150 µl proteinase K buffer (Roche Diagnostics) and extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), and the immunoprecipitated RNA was detected via RT-qPCR as aforementioned.
Proteinase k buffer
Manufactured by Roche
Proteinase K buffer is a solution used to facilitate the activity of the enzyme Proteinase K. Proteinase K is commonly used in molecular biology and biochemistry applications to digest and inactivate proteins. The buffer solution provides an optimal environment for Proteinase K to effectively perform its proteolytic function.
Automatically generated - may contain errors
Lab products found in correlation
Ptc 200 thermocycler, by Bio-Rad (2 mentions)
Ez magna rip kit, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Pbs buffer, by Sangon (1 mentions)
Anti immunoglobulin g anti igg, by Abcam (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Proteinase k, by Roche (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
Anti cpeb4 antibody, by Abcam (1 mentions)
Taq polymerase buffer, by Promega (1 mentions)
4 protocols using proteinase k buffer
1
Argonaute-2 RNA Immunoprecipitation
2
CPEB4 Crosslinking and RNA Immunoprecipitation
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Cpeb4–/– and WT primary BMDMs, untreated or stimulated for 9 hr with LPS (10 ng/ml), were cultured in DMEM supplemented with 10% FBS, rinsed twice with 10 ml PBS, and incubated with PBS 0.5% formaldehyde for 5 min at room temperature under constant soft agitation to crosslink RNA-binding proteins to target RNAs. The crosslinking reaction was quenched by addition of glycine to a final concentration of 0.25 M for 5 min. Cells were washed twice with 10 ml PBS, lysed with a scraper and RIPA buffer (25 mM Tris-Cl pH 7.6, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 100 mM EDTA, 150 mM NaCl, protease inhibitor cocktail, RNase inhibitors), and sonicated for 10 min at low intensity with Standard Bioruptor Diagenode. After centrifugation (10 min, max speed, 4°C), supernatants were collected, precleared, and immunoprecipitated (4 hr, 4°C, on rotation) with 10 μg anti-CPEB4 antibody (Abcam), and 50 μl Dynabeads Protein A (Invitrogen). Beads were washed four times with cold RIPA buffer supplemented with protease inhibitors (Sigma-Aldrich), resuspended in 100 μl Proteinase-K buffer with 70 μg Proteinase-K (Roche), and incubated for 60 min at 65°C. RNA was extracted by standard phenol-chloroform, followed by Turbo DNA-free Kit (Ambion) treatment.
3
Microdissection and Amplification of Spinach Chromosomes
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The X/Y chromosome is the largest submetacentric chromosome (Ellis et al. 1960; Deng et al. 2013 (link)). The microdissection of the largest chromosome in spinach was carried out according to the procedures described by Deng et al. (2013) (link). Initially, the largest chromosome in spinach was identified based on its size and was microdissected using a glass needle that was fixed to the arm of a Leitz micro-operation instrument on an inverted phase-contrast microscope (Olympus 1 M, Japan). Ten chromosomes were isolated. The microdissected chromosome was collected into an Eppendorf tube (0.2 mL) and separately digested with proteinase K buffer at 0.5 mg/mL (Roche, Indianapolis) in 1× Taq polymerase buffer (Promega, Madison). The isolated chromosomal DNA was incubated in a proteinase K solution at 37 °C for 2 h. Proteinase K was then inactivated at 90 °C for 10 min. Then, the chromosome DNA was amplified by DOP-PCR in a PTC-200 thermocycler (MJ Research, Watertown, MA, USA). To obtain high-concentration DNA products for genomic subtraction library construction, the DOP-PCR products were amplified from X and Y chromosome by two rounds of recursive enrichment based on previous studies (primer sequence: CCGACTCGAGNNNNNNATGTGG) (Deng et al. 2013 (link)).
4
Spinach Chromosome Microdissection and DOP-PCR
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The X/Y chromosome is the largest submetacentric chromosome (Ellis et al. 1960; (link)Deng et al. 2013) (link). The microdissection of the largest chromosome in spinach was carried out according to the procedures described by Deng et al. (2013) (link). Initially, the largest chromosome in spinach was identified based on its size and was microdissected using a glass needle that was fixed to the arm of a Leitz micro-operation instrument on an inverted phase-contrast microscope (Olympus 1 M, Japan). Ten chromosomes were isolated. The microdissected chromosome was collected into an Eppendorf tube (0.2 mL) and separately digested with proteinase K buffer at 0.5 mg/mL (Roche, Indianapolis) in 1× Taq polymerase buffer (Promega, Madison). The isolated chromosomal DNA was incubated in a proteinase K solution at 37 °C for 2 h. Proteinase K was then inactivated at 90 °C for 10 min. Then, the chromosome DNA was amplified by DOP-PCR in a PTC-200 thermocycler (MJ Research, Watertown, MA, USA). To obtain high-concentration DNA products for genomic subtraction library construction, the DOP-PCR products were amplified from X and Y chromosome by two rounds of recursive enrichment based on previous studies (primer sequence: CCGACTC-GAGNNNNNNATGTGG) (Deng et al. 2013) (link).
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