LC3 containing complexes were isolated by immunoprecipitation [Pierce Direct IP Kit (Thermo Scientific 26148)]. Ten micrograms of Rabbit polyclonal Ab to LC3 (Abcam 48394) and MOPC-21 purified immunoglobulin (Sigma M5284) were used for coupling to AminoLink Plus Coupling Resin. Columns were incubated on a rotator at room temperature for 120 min to resuspend the slurry. After 2 h of incubation, resin was washed six times with wash buffer. ARPE19 cells were seeded on 100× 100 mm plates a few days before treatment. Cells were 90 % confluent for experiments. To starve cells, we used medium containing 0.5 % FBS overnight at 37 °C and 5 % CO2. To treat cells with POS, 30,000.00 particles were used for 100×100 mm plates for 3 h in incubator at 37 °C and 5 % CO2. Five hundred microliters of lysis buffer was used for both treatments with 1000 ug of protein from each treatment used for immunoprecipitation. After samples were added to antibody-coupled resin, columns were incubated with shaking overnight at 4 °C. Bound proteins were eluted with elution buffer according to manufacturer’s protocol [Pierce Direct IP Kit (Thermo Scientific 26148)].
Pierce direct ip kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce Direct IP Kit is a laboratory product designed for the direct immunoprecipitation of target proteins from complex biological samples. The kit provides the necessary reagents and protocols to facilitate the efficient capture and isolation of proteins of interest for further downstream analysis.
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Lab products found in correlation
P3391 250mg, by Merck Group (2 mentions)
Complete protease inhibitor, by Thermo Fisher Scientific (2 mentions)
Mab1607, by R&D Systems (2 mentions)
Aminolink resin, by Thermo Fisher Scientific (2 mentions)
Anti flna 2c12, by Thermo Fisher Scientific (2 mentions)
Anti flna 3f4, by Thermo Fisher Scientific (2 mentions)
Anti flna 6e3, by Thermo Fisher Scientific (2 mentions)
Ab80579, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Biotinylated pna lectin, by Vector Laboratories (1 mentions)
Precast sds page gel, by Bio-Rad (1 mentions)
Pna lectin, by Merck Group (1 mentions)
α neuraminidase from clostridium perfringens, by Merck Group (1 mentions)
Scn5a, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Roche (1 mentions)
Anti vcl, by Merck Group (1 mentions)
Cell lysis buffer, by Roche (1 mentions)
Cell fractionation kit, by Cell Signaling Technology (1 mentions)
56 protocols using pierce direct ip kit
1
LC3 Immunoprecipitation Isolation and Analysis
2
Immunoprecipitation of SynGAP in Mouse Cortex
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IPs were performed on total protein extracts from cortical samples at different ages. All IPs were performed at a protein concentration of 8 mg/ml. All steps were performed at 4°C in an orbital agitator (Stuart). The following amounts of protein were used for each IP: 9 mg for PND0/1 (n of mice = 24), 16 mg for PND11 (n of mice = 15), 8 mg for PND21 (n of mice = 3), and 7 mg for PND56 (n of mice = 3) samples. IPs were performed as described by the kit manufacturer (26,147, Pierce® Direct IP Kit; Thermo Fisher Scientific). A sepharose resin (Sigma P3391‐250MG) was washed four times with conditioning buffer (50 mM Tris pH 7.4). Next a sample pre‐clearing step was performed by mixing it with washed resin for 2 hr at 4°C. Pre‐cleared sample was mixed with an anti‐SynGAP antibody recognizing an epitope common to all its isoforms (5540S; Cell Signaling Technology [RRID:AB_10695900]) at a 1:15 (v:v) ratio ON. Each 200 μL of pre‐cleared sample were incubated with 7.5 μL of A sepharose resin during 3 hr. A 100 g centrifugation step in a column was performed to recover the resin. Resin was washed three times with dialysis buffer and once with conditioning buffer. Bound protein was eluted with 15 μL of the acidic elution buffer from the kit during 10 min.
3
Co-Immunoprecipitation of K13 Interactors
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Co-IP studies were performed using the Pierce Direct IP kit (Thermo Fisher). Briefly, parasites were extracted from infected erythrocytes as described above, resuspended in Pierce IP Lysis Buffer supplemented with 1× Halt Protease and Phosphatase Inhibitor Cocktail and 25U Pierce Universal Nuclease, and lysed on ice for 10 minutes with frequent vortexing. Samples were centrifuged at 14,000 rpm for 10 min at 4°C to pellet cellular debris. Supernatants were collected and protein concentrations were determined using the DC protein assay kit (Bio-Rad). IPs were performed used 500 μg of lysate per test sample. A mix of K13 E3 and D9 mAbs (2.5 μg each per test sample) was used for IP. Antibody coupling to IP columns, IP, and elution steps were performed according to Pierce instructions. Eluates were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify immunoprecipitated proteins.
4
HOMER3 Immunoprecipitation and ST Antigen Analysis
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A Pierce™ Direct IP Kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions to selectively immunoprecipitate HOMER3 from membrane protein lysates using a rabbit polyclonal anti-HOMER3 antibody (PA5–59383, Thermo Fisher Scientific). The isolated glycoproteins were then run on 4–20% precast SDS-PAGE gels (Bio-Rad), transferred into nitrocellulose membranes and screened for HOMER3 and ST antigens expressions by western blot using the above-mentioned anti-HOMER3 antibody and a biotinylated PNA lectin (Vector laboratories), respectively. ST antigens detection by PNA lectin was preceded by overnight α-neuraminidase from Clostridium perfringens (Sigma-Aldrich) in membrane digestion 0.1 U/mL at 37 °C.
5
Immunoprecipitation and Western Blot Analysis
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Adult mouse heart was dissected and homogenized by 10 strikes with a polytron probe in ice-cold RIPA buffer (Roche). Cell extracts were prepared by solubilizing 107 cells in 1 ml of cell lysis buffer (Roche) for 10 min at 4 °C. After brief sonication, the homogenates or cell lysates were transferred to a 1.5 ml tube and rotated for 1 hour at 4 °C, then centrifuged for 10 min at 10,000 g at 4 °C and the supernatant was transferred to a new tube for protein quantification and immunoprecipitation. Immuno-precipitations were performed with Pierce direct IP kit (Thermo Fisher Scientific Inc., #26148), which immobilized 25 μg VCL (Invitrogen, #MA5–11690) or 25 μg SCN5A (Invitrogen, #PA5-34190) antibodies on agarose-resin support directly to improve specificity. Homogenate (1.0 mg/reaction) was mixed with immobilized antibody-agarose resin complex at 4 °C overnight. After washing to remove non-bound (presumably undesired) components of the sample, the antigen is recovered by dissociation from the antibody-agarose resin complex with elution buffer supplied in the kit. The immunoprecipitated samples were analyzed by Western blotting by probing with anti-SCN5A (Sigma, #SAB2107930) or anti-VCL (Sigma, #V4139).
6
Monocyte-Der p1 Binding Assay
7
Borrelia burgdorferi Protein Co-IP
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Co-IP experiments were performed using the Pierce Direct IP Kit (Thermo Scientific, Rockford, IL). Briefly, B. burgdorferi B31 cells were grown to mid-log phase (5 × 107 organisms ml−1) in complete BSK-II medium before whole-cell lysates were prepared as previously described (Lenhart et al., 2012 (link)). 500 µl of final lysates were then mixed with AminoLink resin with conjugated BB0794, BamA, or GST antibodies following the manufacturer’s instructions (Thermo Scientific). The lysate and resin mixture was rotated end over end at 4°C overnight before being washed. Proteins bound to the resin were eluted by adding 50 µl of final sample buffer containing β-mercaptoethanol and boiled for seven minutes before SDS-PAGE and immunoblot analysis.
8
PAX8 Immunoprecipitation and TEAD Analysis
9
Co-Immunoprecipitation of DJ1-Interacting Proteins
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Immunoprecipitation (IP) was performed with a Pierce Direct IP kit (Thermo Fisher Scientific Inc. Rockford, IL, USA) as per the manufacturer's instructions. Specifically, 20 microgram of anti-DJ1 antibody (1:250; Santa Cruz Biotechnology) was used to precipitate the respective proteins. Rabbit IgG was used as a control. After elution of the proteins, all samples were separated by 10% SDS-PAGE, followed by immunoblotting with anti-DJ1 antibody (Santa Cruz Biotechnology). To identify DJ1-interacting partner proteins in the liver, Co-IP was performed with slight modifications as outlined by Tsai et al. 22 (link). Tissue lysates (pooled sample of six rats per group) containing 300 mg of protein were incubated with 15 μl of anti-DJ1 antibody (Santa Cruz Biotechnology) at 4°C for 5 hrs on a rotary shaker, after which 20 μl of A/G PLUS agarose conjugate suspension (Santa Cruz Biotechnology) was added and allowed to mix at 4°C for 12 hrs on a rotary shaker. After centrifugation at 4000 × g for 5 min. at 4°C, the supernatant was collected and the beads were washed three times with RIPA buffer (Sigma-Aldrich). Then, 2× Laemmli buffer was added and boiled for 5 min. For the negative control sample, control rabbit and rat IgGs (Santa Cruz Biotechnology) were used. After Co-IP, all samples were separated by 10% SDS-PAGE and then silver-staining.
10
Muscle Mitochondrial DNA and Protein Analysis
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RNA was isolated from gastrocnemius muscle using Trizol (Invitrogen) and further purified using RNeasy mini columns (Qiagen). Quantitative PCR analysis was performed on a LightCycler 480II (Roche) using iQ SYBR Green Supermix (Biorad). For mitochondrial DNA quantitation, mitochondrial and genomic DNA was isolated from gastrocnemius muscle after Proteinase K and RNAse A digestion followed by phenol-chloroform extraction. Quantitative PCR analyses were performed using mitochondrial and genomic specific primers.
Skeletal muscle protein homogenates were prepared following standard procedures. The antibodies used were against SIRT1 N-term (Millipore, #07-131), actin (Chemicon, MAB1501), AMPKα (Cell Signaling, #2603), phospho-AMPKα (Cell Signaling, #2531), p70 S6 Kinase (Cell Signaling, #2708), phospho-p70 S6 Kinase (Cell Signaling, #9205), Gapdh (Sigma, G9545), PGC1 (Santa Cruz, sc-13067), acetylated lysine (ImmuneChem, ICP0380). The immunoprecipitation was performed using the Pierce Direct-IP Kit (Thermo Scientific) according to manufacturer's instructions.
NAD+ was measured in freshly isolated gastrocnemius muscle using EnzyChrom kit from BioAssay Systems following the manufacturer's protocol.
Skeletal muscle protein homogenates were prepared following standard procedures. The antibodies used were against SIRT1 N-term (Millipore, #07-131), actin (Chemicon, MAB1501), AMPKα (Cell Signaling, #2603), phospho-AMPKα (Cell Signaling, #2531), p70 S6 Kinase (Cell Signaling, #2708), phospho-p70 S6 Kinase (Cell Signaling, #9205), Gapdh (Sigma, G9545), PGC1 (Santa Cruz, sc-13067), acetylated lysine (ImmuneChem, ICP0380). The immunoprecipitation was performed using the Pierce Direct-IP Kit (Thermo Scientific) according to manufacturer's instructions.
NAD+ was measured in freshly isolated gastrocnemius muscle using EnzyChrom kit from BioAssay Systems following the manufacturer's protocol.
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