A photomultiplier instrument for chemiluminescent signal detection was developed by our laboratory. Chemiluminescent immunoassay instrument (Robust i1000) was provided by Hangzhou AiKang company. The incubation procedures were carried out using a constant temperature incubator. A magnetic separation device was purchased from Thermo Fisher Scientific (Invitrogen). Human liver fatty acid‐binding protein (L‐FABP) in its pure form used in this study was purchased from Sino biological (Sino Biological Inc.). Rabbit anti‐human L‐FABP polyclone antibody pair including detection and capture antibody was provided by Hangzhou Diag Biotechnology Co., Ltd. (China). Streptavidin‐modified paramagnetic particles were purchased from Thermo Fisher Scientific (Invitrogen). N‐hydroxysuccinimide biotin, acridinium ester, bovine serum albumin (BSA), phosphate‐buffered saline (PBS), Tween‐20, dimethylsulfoxide (DMSO), and anhydrous dimethyl formamide (DMF) were purchased from Sigma‐Aldrich Ltd (USA). We prepared hydrogen peroxide and sodium hydroxide reagent. Washing buffer solution was made by PBS containing 1 mg/ml Tween‐20 and 1 mg/ml BSA. Double‐distilled water was prepared using water purified with an ultrapure water system (Zhejiang university second affiliated hospital). All other chemicals were standard commercial products of analytical reagent grade.
Magnetic separation device
Manufactured by Thermo Fisher Scientific
The Magnetic separation device is a laboratory instrument used to separate magnetic particles or beads from a liquid suspension. It generates a strong magnetic field to attract and hold the magnetic particles, allowing the non-magnetic components to be easily removed or collected. The core function of this device is to enable efficient separation and isolation of target analytes or cells for further downstream processing or analysis.
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Lab products found in correlation
Protein a g magnetic bead, by Thermo Fisher Scientific (4 mentions)
Washing buffer, by Thermo Fisher Scientific (3 mentions)
Anti rabbit igg antibody, by Cell Signaling Technology (2 mentions)
Hdac6 antibody, by Proteintech (2 mentions)
Anhydrous dimethylformamide dmf, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ab245539, by Abcam (1 mentions)
Anti rad21 antibody, by Cell Signaling Technology (1 mentions)
Protein a g magnetic beads, by Cell Signaling Technology (1 mentions)
Rad21 antibody, by Cell Signaling Technology (1 mentions)
Ab196026, by Abcam (1 mentions)
Bs 6982r, by Bioss Antibodies (1 mentions)
Ab275378, by Abcam (1 mentions)
Ab181053, by Abcam (1 mentions)
Protein a g magnetic beads, by Abcam (1 mentions)
H3k9ac antibody, by Active Motif (1 mentions)
Fus antibody, by Proteintech (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Protein a g magnetic beads, by Proteintech (1 mentions)
10 protocols using magnetic separation device
1
Chemiluminescent Immunoassay Instrument Development
2
ChIP-qPCR Analysis of EP300 Binding on GPX4 Promoters
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In brief, KG1a cells were crosslinked by 1% formaldehyde for 15 min at RT and then stopped by Glycine. Next, KG1a cells were lysed by sonication to shear DNA. Subsequently, 25 mg DNA chromatin sample was adjusted to a total volume of 500 mL in 450 ml of the dilution buffer contained protease inhibitors. Chromatin samples were then incubated with 1 μg EP300 antibody (#ab275378, Abcam) or anti-rabbit IgG (Cell Signaling Technologies, Danvers, MA, USA) and incubated with protein A/G magnetic beads overnight at 4 °C with gentle rotation. After the overnight incubation, magnetic beads were collected by magnetic separation device (Thermo Fisher Scientific) and cleaned. Next, immunoprecipitated DNAs were eluted with 100 μL elution buffer contained Proteinase K at 62 °C for 2 h. Then DNAs were purified and dissolved in the elution buffer. Finally, chromatin DNAs were analyzed by PCR and qRT-PCR. Primers used were listed as followed: GPX4 promoter 1 forward: 5′-CTGGGCAACACAGCAAGA-3′, reverse: 5′-GGCCAGACAACCTGAGAATAC-3′; GPX4 promoter 2 forward: 5′- CATGCGCAGTCGCCAAC-3′, reverse: 5′-AGACGCGTCGGTGTTGAG-3′; GAPDH promoter forward: 5′-AAAAGCGGGGAGAAAGTAGG-3′, reverse: 5′-AAGAAGATGCGGCTGACTGT-3′.
3
Investigating NIPBL-RAD21-EZH2 Interactions
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H1299 and H1650 cells were lysed in nondenaturing lysis buffer. Next, the supernatant of the cell lysate was precleaned by protein A/G magnetic beads (Thermo Fisher Scientific) for 2 h at 4 °C. Subsequently, approximately 300 μg of protein sample was incubated overnight at 4 °C with 1 μg of anti-NIPBL antibody (#ab245539, Abcam) or anti-RAD21 antibody (#4321, Cell Signaling Technology) and 25 μL of protein A/G magnetic beads. The next day, the protein A/G magnetic beads were collected using a magnetic separation device (Thermo Fisher Scientific), and the precipitated complexes were cleansed with washing buffer (Thermo Fisher Scientific). Finally, the bound proteins were analyzed by Western blotting using KDM6B antibody (#ab38113, Abcam) or EZH2 antibody (#21800-1-AP, Proteintech). Rabbit IgG was used as the negative control.
For competitive Co-IP, different dosages of NIPBL expression vector (0.5 μg, 1 μg, and 2 μg) was transfected into control H1299 or H1650 cells and then the interaction RAD21 and NIPBL or EZH2 was evaluated by Co-IP using anti-RAD21 antibody (#4321, Cell Signaling Technology) as above described.
For competitive Co-IP, different dosages of NIPBL expression vector (0.5 μg, 1 μg, and 2 μg) was transfected into control H1299 or H1650 cells and then the interaction RAD21 and NIPBL or EZH2 was evaluated by Co-IP using anti-RAD21 antibody (#4321, Cell Signaling Technology) as above described.
4
Chromatin Immunoprecipitation Assay Protocol
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In brief, H1299 and H1650 cells were crosslinked by 1% formaldehyde for 15 min at RT before the reaction was stopped by adding 1.375 M glycine. Next, the cells were suspended in lysis buffer and sonicated to shear the DNA. The insoluble material was removed by centrifugation. A 25 mg DNA chromatin sample was adjusted to a total volume of 500 mL in 450 mL of dilution buffer containing protease inhibitors. The chromatin samples were then incubated with 1 μg of NIPBL antibody (#ab245539, Abcam), RAD21 antibody (#4321, Cell Signaling Technology, Danvers, MA, USA), H3K27me3 antibody (#21800-1-AP, Proteintech, Rosemont, IL, USA), enhancer of zeste 2; catalytic subunit of polycomb repressive complex 2 (EZH2) antibody (#21800-1-AP, Proteintech) or anti-rabbit IgG antibody (Cell Signaling Technologies) and incubated with protein A/G magnetic beads overnight at 4 °C with gentle rotation. Magnetic beads were collected by a magnetic separation device (Thermo Fisher Scientific) and cleaned using washing buffer. Subsequently, immunoprecipitated DNAs were eluted with 100 μL elution buffer containing Proteinase K at 62 °C for 2 h. The DNAs were then purified using the spin columns and dissolved in the elution buffer. Finally, chromatin DNA was analyzed by PCR or qPCR.
5
Rapid Affinity Purification (RAP) of Proteins
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RAP was performed by the RAP Kit (Hechuang Biotechnology Co., Ltd.). Briefly, HUVECs were cross-linked by 1% formaldehyde for 10 min at RT and then stopped by glycine. Next, HUVECs were lysed by pre-chilled lysis buffer containing protease inhibitor and RNase inhibitor. Shear DNA was removed using DNase salt stock with DNase for 10 min at 37°C. Subsequently, probes and streptavidin magnetic beads were mixed in 0.5 ml of 1× hybridization buffer for 30 min at RT. Then, cell lysis without DNA was added into 1× hybridization buffer and incubated with probes at 45°C for 90–180 min. Following the incubation with probes, streptavidin magnetic beads were collected using a magnetic separation device (Thermo Fisher Scientific), and precipitated complexes were cleaned five times by washing buffer at 35°C. Next, immunoprecipitated proteins were eluted with elution buffer and analyzed by WB using HDAC6 antibody (1:300, Proteintech).
6
Immunoprecipitation of MET, His, and L-lactyl lysine
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KAS-6/1 and U266 cells were lysed in a non-denaturing lysis buffer, and then the supernatant of the cell lysate was precleaned by protein A/G magnetic beads (Thermo Fisher Scientific) for 2 h at 4℃. Subsequently, approximately 300 µg of protein sample was incubated with 1 µg of MET antibody (#ab216330, Abcam), His antibody (#66005-1-Ig, Proteintech), or L-lactyl lysine antibody (#PTM-1401RM, PTM-BIO, Hangzhou, Zhejiang, China) and 30 µL of protein A/G magnetic beads overnight at 4 °C. The following day, the protein A/G magnetic beads were collected using a magnetic separation device (Thermo Fisher Scientific), and the precipitated complexes were cleaned. Finally, the bound proteins were analyzed by WB. Rabbit or Mouse IgG served as the negative controls.
7
Immunoprecipitation of G-CSF and EP300
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KG1a cells were lysed by the non-denaturing lysis buffer. Next, the supernatant of cell lysis was pre-cleaned with protein A/G magnetic beads (Thermo Fisher Scientific) for 2 h at 4 °C. Subsequently, about 300 μg of protein were incubated with 1μg G-CSF antibody (#ab181053, Abcam) or EP300 antibody (#ab275378, Abcam) and 25 microliters of protein A/G magnetic beads for immunoprecipitation at 4 °C overnight. Following the incubation with antibody and protein A/G magnetic beads, protein A/G magnetic beads were collected using magnetic separation device (Thermo Fisher Scientific), and precipitated complexes were cleansed by washing buffer (Thermo Fisher Scientific). Finally, bound proteins were analyzed by WB using anti-ELANE (1:500, # bs-6982R, Bioss) or anti-TPO (1:500, # ab196026, Abcam). Rabbit IgG was used for negative control.
8
ChIP Assay for Histone Modifications
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ChIP was assessed by the ChIP Kit obtained from Hechuang Biotechnology Co., Ltd. In brief, HUVECs were cross-linked by 1% formaldehyde for 15 min at RT and then stopped by glycine. Next, HUVECs were lysed by sonication to shear DNA. Insoluble material was removed from cell lysis by centrifugation. DNA chromatin sample (25 mg) was adjusted to a total volume of 500 ml in 450 ml of the dilution buffer with protease inhibitors. Chromatin samples were then incubated with 1 μg of HDAC antibody (Proteintech), H3K9ac antibody (Active Motif), or anti-rabbit IgG antibody (Cell Signaling Technologies) and incubated with protein A/G magnetic beads overnight at 4°C with gentle rotation. Magnetic beads were collected by magnetic separation device (Thermo Fisher Scientific) and cleaned using washing buffer. Subsequently, immunoprecipitated DNAs were eluted with 100 μl of elution buffer containing Proteinase K at 62°C for 2 h. Then, DNAs were purified utilizing the spin columns and dissolved in the elution buffer. Finally, chromatin DNAs were analyzed by PCR and qPCR. Primers used for ChIP PCR and qPCR are listed in Table 3 .
9
FUS-Mediated HDAC6 Immunoprecipitation
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HUVECs were lysed by the non-denaturing lysis buffer containing protease inhibitor (Sigma, United States). Next, the supernatant of cell lysis was pre-cleaned with protein A/G magnetic beads (Thermo Fisher Scientific, United States) for 2 h at 4°C. Subsequently, about 300 μg of protein was incubated with 1 μg of FUS antibody (Proteintech, United States) and 25 μl of protein A/G magnetic beads for immunoprecipitation at 4°C overnight. Following the incubation with FUS antibody and protein A/G magnetic beads, protein A/G magnetic beads were collected using a magnetic separation device (Thermo Fisher Scientific), and precipitated complexes were cleansed by washing buffer (Thermo Fisher Scientific). Finally, bound proteins were analyzed by WB using HDAC6 antibody (1:300, Proteintech). Rabbit IgG was used for negative control.
10
Chromatin Immunoprecipitation (ChIP) Protocol
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A ChIP kit obtained from Thermo Fisher Scientific was utilized to assess ChIP. In brief, C2C12 cells were cross-linked by 1% formaldehyde at RT for 15 min and then stopped by glycine. Then, C2C12 cells were sonicated to shear DNA. Next, 25 mg DNA chromatin sample was diluted by 450 mL dilution buffer with protease inhibitors. Subsequently, chromatin samples were incubated with 1 μg Myf5 antibody (Abcam)/antirabbit IgG antibody (Abcam) and magnetic protein A/G beads at 4°C with gentle rotation overnight. After incubation, the magnetic beads were collected using magnetic separation device (Thermo Fisher Scientific) and cleaned. Subsequently, 100 μL elution buffer containing proteinase K was utilized to elute immunoprecipitated DNAs at 62°C for 2 h. Then, the immunoprecipitated DNAs were purified using the spin columns. Finally, chromatin DNAs were analyzed by qPCR. Primers used for ChIP-qPCR in the present study were as follows: Myog ChIP1 forward: 5′-CTGTTGCCCGTGCCGGAGCG-3′, reverse: 5′-AGAATTTGGCCAGATGCAGTG-3′; Myog ChIP2 forward: 5′-GCCCAAACTTCATGATGTCTC-3′, reverse: 5′-AAGAATTTTCCAGGCAGGCC-3′; Myog ChIP3 forward: 5′-CGTCTCCCCAATACGATGTTATG-3′, reverse: 5′-CTCCCCTCCAAGAAAGGGCCAC-3′; Myog ChIP4 forward: 5′-ATCATGGTTCATTGGCAGCC-3′, reverse: 5′-CAGGCACAGTGACTCATGCC-3′.
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