Co-IP assays were carried out using Pierce crosslink immunoprecipitation kits (Thermo Scientific, Rockford, IL, USA) as per the manufacturer's instructions. Cells were treated with 1 ml of extraction buffer (10 mmol/l HEPEs, pH7.5, 100 mmol/l NaCl, 1 mmol/l EDTA, 10% Glycerol, 0.5% Triton X-100 and 5 μmol/l MG132). Co-IP procedures were performed at 4°C unless otherwise indicated, using a Pierce spin column which can be capped and plugged with a bottom plug for incubation or unplugged to remove the supernatant by centrifugation at 1000 g for 1 minute. The binding of the first antibody to protein A/G agarose was performed with the protocol described in Pierce crosslink immunoprecipitation kits (Thermo Scientific, Rockford, IL, USA) with slight modification. For co-IP experiment without using DSS crosslinking, the protein A/G agarose was incubated anti-FSCN1 antibody at 25°C for 1 h on a mixer, followed by incubation with 600 μl pre-cleared lysate overnight. The immunoprecipitated products were washed with the washing buffer five times and eluted with 2X Laemmli buffer at 100°C for 10 min. The cap of the spin column was loose to avoid overpressure and leakage from the bottom when boiling. The eluting complex was subjected to SDS-PAGE separation for Western blot.
Pierce crosslink immunoprecipitation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The Pierce Crosslink Immunoprecipitation Kit is a laboratory tool designed to facilitate the crosslinking of antibodies to beads or resins for immunoprecipitation experiments. It provides the necessary reagents and instructions to perform this procedure.
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Lab products found in correlation
Aprotinin, by Merck Group (5 mentions)
Leupeptin, by Merck Group (5 mentions)
Protein l agarose beads, by Thermo Fisher Scientific (4 mentions)
Bioruptor plus, by Diagenode (4 mentions)
Pierce supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Mouse igg, by Thermo Fisher Scientific (2 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (2 mentions)
Protease inhibitor cocktail mixture, by Merck Group (2 mentions)
Anti flag m2 affinity gel, by Merck Group (2 mentions)
Pierce co immunoprecipitation kit, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Protease and phosphatase inhibitor, by Roche (2 mentions)
Smad2 overexpression plasmid, by Addgene (2 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Sample buffer, by Thermo Fisher Scientific (2 mentions)
Control agarose resin slurry, by Thermo Fisher Scientific (2 mentions)
Rabbit anti flag antibody, by Abcam (2 mentions)
Reducing agent, by Thermo Fisher Scientific (2 mentions)
Peroxidase coupled anti mouse secondary antibody, by Thermo Fisher Scientific (2 mentions)
63 protocols using pierce crosslink immunoprecipitation kit
1
Co-Immunoprecipitation Assay Protocol
2
Crosslink Immunoprecipitation Protocol
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Immunoprecipitation from protein preparations were performed using the Pierce Crosslink Immunoprecipitation Kit (Pierce Biotechnology, Rockford, IL) according to the instruction of manufacturer. Briefly, cell lysates were incubated with antibody-cross-linked Pierce Protein A/G Plus Agaros overnight at 4 °C. The immunoprecipitates were eluted and subjected to Western blot analysis.
3
Amygdala and Hippocampus Synaptosomal Analysis
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Immediately after the termination of the CH test (for both Day-1 and Day-14 groups), the animals were anesthetized using chloral hydrate solution (400mg/kg). Subsequent decapitation and microdissection of the brain to obtain amygdala and dorsal hippocampus (dHC) were performed using the rat brain stereotaxic coordinates (Paxinos and Watson, 2013 ). The brain regions were isolated into microcentrifuge tubes, immersed in liquid nitrogen to prevent degradation, and stored at -80°C till further use. A crude synaptosomal prep was made from these brain tissues, and subsequent Western blotting was performed using standard procedures. A uniform concentration (10–20 µg) of protein was diluted in Laemmli sample buffer (6X) and heated for 20 minutes at 70°C (Chinese hamster ovary [CHO] cell extracts were denatured for 5 minutes to prevent aggregation that occurred on longer duration of heating).
Antibodies (45 µg/reaction) were covalently crosslinked onto protein A/G resin according to the manufacturer’s instructions (Pierce Crosslink Immunoprecipitation Kit, Pierce Biotechnology, Rockford, IL), and immunoprecipitation studies were conducted using standard protocols.
Antibodies (45 µg/reaction) were covalently crosslinked onto protein A/G resin according to the manufacturer’s instructions (Pierce Crosslink Immunoprecipitation Kit, Pierce Biotechnology, Rockford, IL), and immunoprecipitation studies were conducted using standard protocols.
4
Enriching Autoantibody Target Antigens
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Target antigens of potential autoantibodies were enriched using the Pierce Crosslink Immunoprecipitation Kit (Pierce Biotechnology, Rockford, United States). Briefly, equal volumes of three serum samples from AMA-positive PBC patients were pooled, resulting in three AMA-positive PBC sample pools. AMA-negative PBC samples were handled identically. All pooled samples were diluted and incubated with protein A/G agarose to capture antibodies. Then, the antibodies were cross-linked to prevent co-elution with antigens.
HiBECs were lysed using the immunoprecipitation lysis and wash buffers provided in the immunoprecipitation kit. Control agarose resin was used to pre-clear the lysate, adsorbing non-specific binding entities. To ensure the formation of target complexes, cleared lysates with excess antigens were incubated with the antibody-agarose complexes. The target antigens were eluted from these complexes, using low-pH elution buffer provided with the kit. Then, 1 M Tris pH 9.5 was added to the eluate to neutralize the pH. The final collections were stored at -80 °C until proteomic analysis.
HiBECs were lysed using the immunoprecipitation lysis and wash buffers provided in the immunoprecipitation kit. Control agarose resin was used to pre-clear the lysate, adsorbing non-specific binding entities. To ensure the formation of target complexes, cleared lysates with excess antigens were incubated with the antibody-agarose complexes. The target antigens were eluted from these complexes, using low-pH elution buffer provided with the kit. Then, 1 M Tris pH 9.5 was added to the eluate to neutralize the pH. The final collections were stored at -80 °C until proteomic analysis.
5
Probing PfHsp90-PfSir2A Interaction
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Co-IP was performed as previously described (17 (link)) using the Pierce Crosslink Immunoprecipitation Kit (Thermo Scientific). For determining the interaction between PfHsp90 and PfSir2A, pull-down was performed by cross-linking anti-PfSir2A, or anti-Hsp90 to Pierce Protein A/G Plus Agarose beads. For the negative control, beads were cross-linked to anti-IgG for performing the pull-down.
6
Coimmunoprecipitation Protocol for β-catenin
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Coimmunoprecipitation was performed using a Pierce Crosslink Immunoprecipitation Kit according to the manufacturer's instructions (ThermoFisher, Waltham, MA). Briefly, 10 μg of β-catenin antibody (Thermo Fisher, MA1–301, 1 mg/ml) or mouse IgG (ThermoFisher, 31903, 5.6 mg/ml) were added to a spin column containing 20 μl of resin slurry in a 2 ml collection tube, and the mix was incubated at room temperature with gentle rotation for one hour. After incubation, the spin columns were centrifuged, and then the antibody-bound resin was rinsed with ×1 coupling buffer three times to remove unbound antibodies. A 2.5 mM disuccinimidyl suberate (DSS) crosslinker solution was added to crosslink the antibody to the resin. Next, 750 μg of protein extract from either GFP/Fb or bcat-GFP/Fb in a total volume of 500 μl was added to the spin column and rotated end-over-end at 4 °C overnight. After incubation, the column was washed three times with ×1 TBS and one time with ×1 conditioning buffer. Then, 50 μl of elution buffer was used to elute the samples from the column. The eluate was boiled in ×1 sample buffer at 95 °C for 5 min for Western blot analysis.
7
Cross-linking Immunoprecipitation for RXR-γ
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CoIP was performed according to the instructions on the cross-linking CoIP kit (Thermo Fisher Scientific). In brief, OPCs and OLGs were grown for 5 DIV and lysed with immunoprecipitation lysis wash buffer (0.025-M Tris, 0.15-M NaCl, 0.001-M EDTA, 1% NP-40, and 5% glycerol; Thermo Fisher Scientific) with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Protein concentration was measured by bicinchoninic acid assay (Thermo Fisher Scientific), and immunoprecipitation was performed following the Pierce cross-link immunoprecipitation kit protocol (Thermo Fisher Scientific). In brief, 10 µg RXR-γ antibodies (ab15518; Abcam) were bound to protein A–coated agarose beads. Between 200 and 600 µg of protein were incubated overnight at 4°C with the antibody. Elution was analyzed by Western blotting.
8
Immunoprecipitation of Connexin-43, AIF, and ETFB
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A total of 0.8–1 mg of protein was used for each pull down reaction with 10 μg of rabbit anti‐connexin‐43 antibody (C6219; Sigma‐Aldrich), mouse anti‐apoptosis‐inducing factor antibody (MA5‐15880; Pierce‐Thermo Scientific, Rockford, IL, USA), rabbit anti‐ETFB subunit beta antibody (17925‐1‐AP; ProteinTech, Manchester, UK), respectively, according the Pierce® Crosslink Immunoprecipitation Kit manufacturer's instructions (26147; Thermo Scientific, Rockford, IL, USA). Total and mitochondrial heart extracts were treated in non‐denaturing conditions with buffers supplied, and protein‐antibody‐agarose beads complexes incubated at 4°C during 16 hrs with an end‐over‐end mixing. After washing, bound proteins were eluted (low pH) and preserved for posterior proteomics analysis and Western blotting.
9
Protein-Protein Interaction Analysis
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The RKO cells were cultured conventionally and transfected with pcDNA6/myc-His B-TAGLN-flag and pcDNA6/myc-His B-flag plasmids. The RKO cells were transfected with pENTER-TAGLN-Flag and pENTER-Flag control plasmids in the validation experiment. Cells were then harvested at 48 h after transfection for further analysis. Antibody immobilization, cell lysis, pretreatment of cell lysate with control agarose resin, immunoprecipitation, immunoprecipitation elution, and immunoblotting analysis were performed in sequence according to the protocol of the Pierce Crosslink Immunoprecipitation Kit (Thermo Fisher Scientific, USA). Anti-flag antibody (10ug, Sigma-Aldrich, USA, for the subsequent mass spectrometry; 1:50, Cell signaling technology, USA, for the validation experiment) and the control rabbit IgG (1:50, Cell signaling technology, USA) were used.
10
Mass spectrometry analysis of SmRho1 immunoprecipitation
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For mass spectrometry analysis, two independent experiments were performed. Adult worms (100 couples) were suspended in 500 μL of lysis buffer (20 mM Tris-HCl pH 7.4, 50 mM NaCl, 5 mM EDTA, 1% Triton and protease inhibitors (20 μM leupeptin (Sigma), 2 μg mL-1 aprotinin (Sigma), 200 μM phenylmethylsulfonyl fluoride (Sigma), crushed with a Dounce homogenizer and sonicated ten times for 30 s (maximum power, Bioruptorplus, Diagenode). After centrifugation at 1000 x G for 10 min at 4°C, immunoprecipitation of SmRho1 was performed using the Pierce Crosslink Immunoprecipitation Kit (Thermo Scientific) according to the manufacturer’s instructions. Briefly, the protein lysate (500 μL) was pre-cleared by incubation with 20 μL of IgG from rat serum crosslinked to protein-L Agarose beads (5 mg of beads, Thermo Scientific) for 2h at 4°C on a rotator. Then, pre-cleared lysate was collected after centrifugation, at 1000 x G for 1 min at 4°C, and incubated overnight at 4°C on a rotator, with 10 μL of anti-SmRho1 antibodies or 10 μL of IgG from mouse serum as a control, bound to protein-L Agarose beads (5 mg of beads, Thermo Scientific).
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