IP assays were performed using the Pierce™ Direct Magnetic IP/Co-IP Kit (Thermo Fisher) according to the manufacturer’s instructions. The cells were washed with ice-cold HBSS, and lysed in 1mL lysis buffer (Invitrogen). Washing equal 50 μL Magnetic Beads (A/G) (Invitrogen) with yeast RNA (Ambion) or IP Lysis/Wash Buffer, the cells were further incubated with 5 μg primary antibody or IgG as negative control (NC) for 1 h. Equal amounts of proteins were incubated with the antibody-coupled magnetic beads for 4 h at 4° C on a rotator. The pellets were washed 3 times with lysis buffer (Invitrogen) and resuspended in Elution Buffer (Invitrogen), followed by Western Blot analysis.
Elution buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Elution buffer is a laboratory reagent used to release or recover molecules, such as proteins or nucleic acids, from a solid phase or chromatography medium. It provides the necessary chemical conditions to displace the bound target analyte from the stationary phase, enabling its collection and further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (6 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (5 mentions)
Washing buffer, by Thermo Fisher Scientific (4 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (4 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Protein g dynabead, by Thermo Fisher Scientific (3 mentions)
Wash buffer, by Thermo Fisher Scientific (3 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (3 mentions)
Binding buffer, by Thermo Fisher Scientific (3 mentions)
Dynabead, by Thermo Fisher Scientific (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Latex agglutination, by Statens Serum Institut (2 mentions)
Pierce co immunoprecipitation kit, by Thermo Fisher Scientific (2 mentions)
Streptavidin magnetic beads, by Thermo Fisher Scientific (2 mentions)
Surebeads protein g magnetic beads, by Bio-Rad (2 mentions)
Q exactive instrument, by Thermo Fisher Scientific (2 mentions)
Control agarose resin, by Thermo Fisher Scientific (2 mentions)
Pierce protein a g magnetic beads, by Thermo Fisher Scientific (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
54 protocols using elution buffer
1
Magnetic Bead-Based Immunoprecipitation Protocol
2
Extraction and Purification of Viral RNA
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Frozen stool samples were thawed on ice, and a 10% stool suspension was created in 500 μL of phosphate-buffered saline. The stool suspension were lysed using a MagNA Lyser (Roche) at 6500 rpm for two 30-second periods, with 30 seconds on a cooling block (4°C) in between to keep samples from degrading because of excessive heat. The lysed suspensions were centrifuged at 8000g for 2 minutes to pellet any debris. Next, 200 μL of the supernatant was added to a 96–deep well plate for processing in a KingFisher Duo Prime system (Thermo Scientific). Each 200-μL aliquot was spiked with 1 μL of the bacteriophage MS2 (American Type Culture Collection), to act as an internal positive control for RNA extraction. If the MS2 cycle threshold (Ct) reading during the RT-qPCR assay was >37, the run was considered invalid and the sample was reprocessed. Carrier RNA (2 μL) (Ambion; Life Technologies) was added to enhance the nucleic acid yield. Viral RNA was extracted from the supernatant according to the manufacturer’s instructions (Invitrogen or Thermo Fisher Scientific) for isolating viral RNA. The viral RNA was eluted into 50 μL of elution buffer (Invitrogen) and stored at −20°C until it was ready for RT-PCR processing.
3
Immunoprecipitation of PARC/H7AP1, ANAPC7, and FZR1
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Immunoprecipitation experiments were performed by incubating 2mg of whole-cell lysates with 2 μg of PARC/H7AP1 (Bethyl, Cat. A300-98A), ANAPC7 (Bethyl, Cat. A302-551A) or FZR1 (Abcam, Cat. ab3242) for 1 hr at 4°C. 40 μL of 50/50 slurry of Dynabeads (Invitrogen) was added to sample and left to incubate for 1 hr at 4°C. Beads were separated by magnet and washed in 1% Triton. Protein was eluted and incubated with Elution Buffer (Invitrogen) and LDS/BME sample buffer at 95°C for 5 min. Immunoprecipitations were analyzed by Western blot or LC-MS/MS (See S1 File for detailed LC-MS/MS methods).
4
Immunoprecipitation and Western Blot
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Cells were rinsed two times in ice-cold PBS and lysates were collected in NP40 buffer (Thermo Fisher) supplemented with protease and phosphatase inhibitor (Thermo Fisher). Protein concentrations were measured using BCA assay (Thermo Fisher) and determined using a standard curve of bovine serum albumin (BSA) serially diluted in PBS. Luminescence was measured on SpectraMax. Equivalent amounts of total protein were added to 50 μL of suspended Protein G Dynabeads (Invitrogen) which were previously pre-cleared and incubated with 4 μg of HA antibody (Biolegend). Lysate was incubated with beads overnight at 4 ˚C. The next day, bead and lysates were washed three times in PBS supplemented with 0.1% Tween-20 (Bio-Rad). 40 μL of elution buffer (Invitrogen) and 1× LDS loading buffer (Invitrogen) was added and samples were boiled at 70 ˚C for 10 min for elute proteins bound to the bead-antibody complex. Samples were then placed on the magnet (Invitrogen) to remove beads and load protein sample using standard Western blot procedures under reducing conditions.
5
Enrichment of EV Subpopulations Using Magnetic Beads
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Samples were enriched for CD63, CD9, and CD81 positive EV using magnetic beads (ThermoFisher 1060D, 10620D, and 10622D, respectively). Briefly, the total EV isolated as above were added to 80 μL of equilibrated, antibody-coated magnetic beads. Non-specific IgG-coated beads were used as a control. EV were bound to beads overnight at 4˚C and beads were washed 3X with 1X PBS. EV were eluted in 100 μL of elution buffer (Invitrogen) or 0.2 M Glycine pH = 2.0 for further analysis. Cell were authenticated by targeted amplification of STR typing loci using Ion Torrent Precision ID GlobalFiler NGS STR Panel and compared against the STR database of the German Collection of Microorganisms and Cell Cultures GmbH.
6
Fecal DNA Extraction Protocol
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Mice were placed in an empty autoclaved glass beaker and one fresh fecal pellet was transferred to a sterile 2 ml round-bottom tube (Nalgene, Rochester, NY) on dry ice and stored at − 80 °C until further analysis. For DNA extraction, lysis buffer was adjusted by fecal weight and up to 800 μl was added per bead-containing tube following the manufacturer’s instructions using Purelink Microbiome DNA Purification Kit (Invitrogen, Carlsbad, CA, USA). Samples were homogenized (30 s) in a bead homogenizer (Thermo Fisher, Waltham, MA) and centrifuged at 14000 g (5 min), and the supernatants were collected in a 1.5 mL microcentrifuge tube (Eppendorf, Hauppauge, NY). Samples were transferred to a spin column for purification and eluted in 100 μl elution buffer (Invitrogen by Thermo Fisher). DNA concentrations were determined by spectrophotometry (Nanodrop, Wilmington, DL). The purified DNA was used for bacterial 16S rRNA qPCR to monitor potential contamination. For bacterial 16S rRNA gene sequencing, DNA of feces from GF mice was extracted using QIAamp DNA Stool Mini kit following the manufacturer’s instructions (Qiagen Ltd., Strasse, Germany) for quality control.
7
Protein-protein interaction assay
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A total of 5 μg mouse anti-His, anti-HA, or anti-Flag monoclonal antibody (CWBiotech) was incubated with 50 μL Dynabeads Protein G (Novex, Thermo Fisher Scientific) for 15 min at room temperature. Then, 400 µL 1:1 mixture of recombinantly expressed importin α2 fragment (IBB-His, Arm-His or carboxyl-terminal–His) and NP-Flag, importin β–HA and NP-Flag, importin β–HA and IBB-His, or HSPG His-tagged five fragments and NP-Flag were added and incubated for 2 h at 4 °C. In another group, 400 µL IBB-His and 400 µL total protein from viruliferous planthoppers in 10 mM PBS buffer (pH 8.0) was added and incubated for 2 h at 4 °C. The total protein from E. coli–expressing empty pET28a was applied in the negative control groups. A total of 5 μg NP monoclonal antibody was first incubated with 50 μL Dynabeads Protein G (Novex) for 30 min at room temperature, after which 400 μL total protein from viruliferous planthoppers in 10 mM PBS buffer (pH 8.0) was added. Approximately 10% of the total protein was reserved as input. Mouse IgG (Merck Millipore) was used as a negative control. After washing three times with washing buffer (Novex), the antibody–protein complex was disassociated from the beads with elution buffer (Novex) for Western blot analysis with anti-His, anti-HA, anti-Flag, or anti-NP monoclonal antibodies or anti–importin α2 polyclonal antibodies.
8
Brain Protein Immunoprecipitation and Analysis
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Immunoprecipitation was performed according to a previous study (Zhao et al, 2021 ). Approximately 15 brains were homogenized using 1× PBS buffer (pH 7.2) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific). Then, 50 μl of Dynabeads Protein G (Novex, Thermo Fisher Scientific) was mixed with 5 μg of anti‐Piwi1 monoclonal antibody or anti‐ Mouse IgG (Merck Millipore) antibody before being incubated with 200 μl of protein extracts for 15 min at room temperature. Approximately 10% of the total protein was reserved as input. After washing three times with washing buffer (Novex), the antibody–protein complex was disassociated from the beads with elution buffer (Novex) for Western blot analysis.
9
Protein-Protein Interaction Assay
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Five micrograms of a mouse anti‐His monoclonal antibody (CWBiotech, Beijing, China) was incubated with 50 μL of Dynabeads® Protein G (Novex®, Thermo Fisher Scientific, Waltham, MA) for 15 min at room temperature. Then, 400 μL of a 1:1 mixture of recombinant Flotillin 1‐Flag and RSV NP‐His, RBSDV P10‐His, SRBSDV P10‐His, RRSV P8‐His, RGSV P5‐His, RGDV P8‐His, RDV P2‐His or RDV P8‐His; a 1:1 mixture of RSV NP‐His and each Flag‐tagged Flotillin 1 fragment (N‐Flag or C‐Flag) or Flotillin 3‐Flag or a 1:1 mixture of Flotillin 1‐His and Flotillin 3‐Flag was added, and incubation was performed for 2 h at 4 °C. In another group, 5 μg of a mouse anti‐Flag monoclonal antibody (CWBiotech) was incubated with 50 μL of Dynabeads® Protein G for 15 min, and then 400 μL of Flotillin 1‐Flag or Flotillin 3‐Flag and 400 μL of the total protein extracted from RSV‐infected rice were added. The total protein from RSV‐infected rice was extracted using a plant protein extraction kit (CWBiotech) following the manufacturers' instructions. Mouse IgG or total protein from E. coli expressing empty pET28a was applied in the control groups. After washing three times with washing buffer (Novex®), the antibody‐protein complex was disassociated from the beads with elution buffer (Novex®) for western blot analysis with anti‐His, anti‐Flag or anti‐NP monoclonal antibodies.
10
Anxa2 and p50 Immunoprecipitation Assay
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Immunoprecipitation was performed as previously described (47 (link)). BV2 cells were grown in 10 cm dishes. After OGD/R, cells were extracted in cold lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, and 0.5% sodium deoxycholate) containing protease inhibitor mixture (Sigma, P8340). Cell lysates were sonicated and centrifuged at 20,000g for 10 min at 4 °C. Cleared lysates were incubated with anti-Anxa2 (Proteintech, 11256-1-AP) or anti-p50 (Cell signaling Technology, 13586) antibodies overnight at 4 °C and then precipitated with protein A/G magnetic beads at 4 °C for 2 h. After incubation, the beads were washed three times with washing buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA, and 0.5% sodium deoxycholate) and eluted with elution buffer (Thermo Fisher Scientific, 21004) at room temperature for 10 min. The eluents were neutralized with 1 M Tris–HCl (pH 8.0) and analyzed by immunoblotting.
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