Cellular lysates were prepared by incubating the cells in NETN buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.2% Nonidet P-40, 2 mM EDTA) in the presence of protease inhibitor Cocktails (Roche) for 20 min at 4 °C followed by centrifugation at 14,000 g for 15 min at 4 °C. For immunoprecipitation, about 500 μg of protein was incubated with control or specific antibodies (1-2 μg) for 12 h at 4 °C with constant rotation; 50 μL of 50% protein G magnetic beads (Thermo Fisher) was then added and the incubation was continued for an additional 2 h. Beads were then washed five times using the lysis buffer. Between washes, the beads were collected by magnetic stand (Thermo Fisher) at 4 °C. The precipitated proteins were eluted from the beads by re-suspending the beads in 2 × SDS-PAGE loading buffer and boiling for 5 min. The boiled immune complexes were subjected to SDS-PAGE followed by immunoblotting with appropriate antibodies.
Magnetic stand
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Magnetic stand is a laboratory equipment used to hold and separate magnetic particles or beads in a sample. It generates a strong magnetic field to capture and retain the magnetic particles, allowing for easy aspiration or removal of the non-magnetic components.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (5 mentions)
Protein g magnetic beads, by Thermo Fisher Scientific (4 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Normal rabbit igg, by Cell Signaling Technology (2 mentions)
Fragmentation reagent, by Thermo Fisher Scientific (2 mentions)
Ribominus, by Thermo Fisher Scientific (2 mentions)
Stratalinker, by Agilent Technologies (2 mentions)
Protein a g bead, by Thermo Fisher Scientific (2 mentions)
Magnetic separation stand, by Thermo Fisher Scientific (1 mentions)
Pierce magnetic rna protein pull down kit, by Thermo Fisher Scientific (1 mentions)
C 1 magnetic beads, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Protein a g magnetic beads, by MedChemExpress (1 mentions)
Np 40 lysis buffer, by Beyotime (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Mouse igg1 kappa, by Thermo Fisher Scientific (1 mentions)
Direct magnetic ip co ip kit, by Thermo Fisher Scientific (1 mentions)
3xflag peptide, by Apexbio (1 mentions)
24 protocols using magnetic stand
1
Immunoprecipitation of Protein Complexes
2
APN Ortholog-B Domain Interaction Analysis
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The interaction between APN orthologs and B domains was further analyzed performing a pull-down assay. Briefly, 200 μl (2 mg) of magnetic beads Dynabeads™ Strep Isolation (ThermoFisher) were washed twice with 300 μL 1X TBS-T (2.4 g Tris-Base/L, 8 g NaCl/L, 0.1 % Tween20) using a magnetic stand (ThermoFisher) before coupling them with 200 μl of B domains in TBS at 0.125 ng/ml. The mixture beads/protein were well mixed and incubated for 1h at room temperature with constant gentle rotation. Beads were then washed two times with 200 μL TBS-T before resuspending them in a solution containing an excess of 10-20X of their calculated KD,app using BLI, of the APN-Fc orthologs and allow binding for 1 hr at RT with gentle rotation after which were washed two times with TBS-T. Flow through, last wash and beads were collected and analyzed in SDS-PAGE gel. Control assays consisted of beads alone, i.e: not coupled to B domains.
3
Verification of circTADA2A-miR-638 Interaction
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The targeted binding effect between circTADA2A and miR-638 was verified by RNA pull-down assay as previously reported (25 (link)). Biotinylated miR-638 probe was purchased from Shanghai GenePharma Co., Ltd. A549 and H1299 cells (1×106) were harvested, lysed by 200 µl Pierce® IP lysis buffer (Thermo Fisher Scientific, Inc.) and sonicated. The pull-down assay was performed using a Pierce™ Magnetic RNA-Protein Pull-Down kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. In brief, 50 µl C-1 magnetic beads (Thermo Fisher Scientific, Inc.) were co-incubated with 50 pmol biotinylated miR-638 or NC probes at 25°C for 30 min to generate probe-coated beads. The beads were harvested by a magnetic stand (Thermo Fisher Scientific, Inc.), then incubated with cell lysate at 4°C for 60 min. The beads were harvested using a magnetic separation stand (Thermo Fisher Scientific, Inc.). The pulled down RNA complexes were eluted and extracted using a RNeasy Mini kit (Qiagen, Inc.), Pulled down circTADA2A and circular nucleolar protein 10 (NOL10) were determined by RT-qPCR, as aforementioned.
4
AMPK-FoxO1 Interaction Detection
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Co‐immunoprecipitation analysis was conducted to detect AMPK‐FoxO1 interaction. In brief, heart tissue lysates were acquired using NP‐40 lysis buffer (Cat No. P0013F, Beyotime). Following protein concentration determination, equal amount of samples (about 500 μg) were incubated with 1.5 μg of primary antibodies at 4°C overnight on a rocker. Protein A/G magnetic beads (Cat No. HY‐K0202, MedChemExpress) were then used to precipitate antibody‐bound proteins. After 4 h precipitation at room temperature, the beads were separated by a magnetic stand (Thermo Fischer Scientific) and washed with ice‐cold PBST solution (0.1% Tween‐20 in PBS). After elution in SDS loading buffer in boiling water for 5 min, eluted proteins were further analysed by western blot.
5
Targeted Protein Immunoprecipitation Analysis
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Co-immunoprecipitation was performed using the Direct Magnetic IP/Co-IP Kit (Thermo Scientific) following the manufacturer’s protocols. Primary antibodies (5 μg/reaction) against Flag (#14,793, CST), ATF6 (sc-166659, Sigma-Aldrich), rabbit IgG (#3900, CST), and mouse IgG1 kappa (14–4714-82, eBioscience) were used respectively to couple to N-hydroxysuccinimide-activated magnetic beads (25 μL/reaction) for 30 min at room temperature. The cells were lysed and extracted using lysis buffer. The protein concentration was then estimated using the Bicinchoninic acid Protein Assay Kit (88,828, Thermo Scientific). Lysate solution with 750 μg of proteins was added to the antibody-coupled magnetic beads and incubated overnight at 4 °C on a shaker (88,881,002, Thermo Scientific) for antigen immunoprecipitation. The immunoprecipitation complex was eluted using a magnetic stand (21,359, Thermo Scientific), and the eluted proteins were electrophoresed and analyzed by western blotting.
6
Magnetic Bead Preparation for ISFET Measurements
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In accordance with the gate size of the ISFET used in this study, magnetic beads with 90 μm diameter were employed in this study. As the magnetic beads were delivered in a low-salt buffer with 20% ethanol, thorough washing was necessary before electrical measurements. Magnetic beads (5 μL) in a stock solution were transferred into a 1.5 mL microcentrifuge tube with 50 μL deionized water and then magnetically captured on the sidewall using a magnetic stand (Thermo Fisher). The supernatant was aspirated, and the magnetic beads were washed three times with 100 μL measurement solutions (1X PBS or other measurement solutions). After washing, the magnetic beads were resuspended in a 50 μL measurement solution. Each 10 μL pretreated solution included 1 μL magnetic beads, that is, the number of magnetic beads was about 100.
7
Immunoprecipitation and Western Blotting
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Cells were lysed in mammalian cell lysis buffer (50 mM Tris-Cl [pH 6.8], 150 mM NaCl, 0.5 % Nonidet P-40, Halt protease inhibitors [Pierce], and 1 mM DTT). To recover total protein (soluble and insoluble), sodium dodecyl sulfate (SDS) was added to a final concentration of 1% (Figure 1B, Supplementary Figure 2F and6A).
Cells were incubated at 4°C for 10 min and then centrifuged at 14,000 rpm for 15 min. The supernatant was removed, and the protein concentration was estimated using the DCA assay (Bio-Rad).
Myc or FLAG magnetic beads in a 1:1 slurry in mammalian cell lysis buffer was added to samples and rotated at 4°C overnight. A magnetic stand (Thermo Fisher) was used to wash the beads three times with lysis buffer. Beads were resuspended in 25 µl SDS sample buffer, boiled briefly, and processed for SDS-PAGE and immunoblotting. FLAG elutions were performed two times with the 250µg/ml 3XFLAG peptide (ApexBio) for 30 minutes at room temperature. Endogenous p97 was further immunoprecipitated (Bethyl) at 4°C overnight.
Beads were washed 3 times in lysis buffer and resuspended in Laemmli buffer and boiled briefly.
Cells were incubated at 4°C for 10 min and then centrifuged at 14,000 rpm for 15 min. The supernatant was removed, and the protein concentration was estimated using the DCA assay (Bio-Rad).
Myc or FLAG magnetic beads in a 1:1 slurry in mammalian cell lysis buffer was added to samples and rotated at 4°C overnight. A magnetic stand (Thermo Fisher) was used to wash the beads three times with lysis buffer. Beads were resuspended in 25 µl SDS sample buffer, boiled briefly, and processed for SDS-PAGE and immunoblotting. FLAG elutions were performed two times with the 250µg/ml 3XFLAG peptide (ApexBio) for 30 minutes at room temperature. Endogenous p97 was further immunoprecipitated (Bethyl) at 4°C overnight.
Beads were washed 3 times in lysis buffer and resuspended in Laemmli buffer and boiled briefly.
8
Magnetic Bead-Based DNA Extraction
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400 ng of human gDNA (Promega, Wisconsin, USA) in 20 µL water was first mixed with binding buffer and silica magnetic particles (Magnetic Suspension G, Qiagen). After the incubation, the liquid was removed by placing the reaction unit on the absorbent pad. The washing process was done by adding the washing buffer to the reaction chamber, and subsequently removing the waste washing buffer through the membrane using the absorbent pad. In the end, 20 µL of water was added to elute DNA from the particle surface. The eluent was retrieved from the top with a pipette. For comparison, DNA was also isolated in the microcentrifuge tube using the same protocol. To exchange liquid in the microcentrifuge tube, the tube was placed on a magnetic stand (Thermo Fisher Scientific, Massachusetts, USA). To evaluate the purity of isolated DNA, the 260/280 and 260/230 ratios were measured using the Nanodrop ND-1000 UV-Vis spectrometer (Thermo Fisher Scientific, Massachusetts, USA). The concentration of the isolated DNA was measured using the PicoGreen assay (Thermo Fisher Scientific, Massachusetts, USA).
9
Immunoprecipitation Assay for Protein Analysis
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Cell lysates were prepared by incubating cells in NETN buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.2% Nonidet P-40, 2 mM EDTA) in the presence of Protease Inhibitor Cocktails (Roche) for 20 min at 4 °C. This step was followed by centrifugation at 14,000×g for 15 min at 4 °C. For IP, B500 mg of protein was incubated with control or specific antibodies (1–2 mg) for 12 h at 4 °C with constant rotation. A total of 50 ml of 50% protein G magnetic beads (Invitrogen) were then added, and incubation was continued for an additional 2 h. Beads were then washed five times using lysis buffer. Between washes, beads were collected by magnetic stand (Invitrogen) at 4 °C. Precipitated proteins were eluted from beads by re-suspending beads in 2 SDS-PAGE loading buffer and boiling for 5 min. Boiled immune complexes were subjected to SDS-PAGE, followed by immunoblot with the appropriate antibodies.
10
Biotin-P-LPK Protein Interactome Capture
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50 μg/ml Biotin-P-LPK (Biotin-P-CON) was incubated with HCT116 at 37 °C for 2 h. Then cells were lysed and centrifuged at 12,000 rpm for 20 min at 4 °C. The above supernatant was then suspended with pretreated magnetic beads overnight at room temperature, and the supernatant was collected for control (as supernatant group). The magnetic beads were eluted once with 200 μl RIPA lysis solution (First elution, as supernatant group 1) and captured with a magnetic stand (Invitrogen, USA), and the magnetic beads were eluted for the second time (Second elution, as supernatant group 2). Subsequently, the magnetic beads were added to 20 μl loading buffer and boiled at 100 °C for 10 min. Finally, the beads-P-LPK-specific binding protein compounds were collected and diluted with ddH2O (Co-IP products). All collected protein complexes were eluted with 50 ml of ddH2O by boiling for 10 min and then subjected to silver staining.
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