Three independent Clpp−/− MEF lines were transfected with each empty vector (NEG), CLPP-WT-FLAG (WT) and a catalytically inactive version with Ser149 mutated to Ala, CLPP-TRAP-FLAG (TRAP) (13 (link)) using the Nucleofector (Lonza, Basel, Switzerland) electroporation kit according to the manufacturer instruction. 72h after transfection, 80% confluent cells were harvested with trypsin, washed twice with PBS and lysed for 45 min in 300 μl IP buffer (Thermo Fisher Scientific, Schwerte). Samples were incubated with 30 μl α-FLAG magnetic beads (Sigma) over night at 4 °C on a rotating wheel. On the following day, beads were washed 4 times with IP buffer and bound proteins were eluted in 70 μl Elution buffer (Thermo Fisher Scientific). Samples were neutralized with 10 μl 1 m Tris/HCl pH 7.5, snap-frozen and stored at −80 °C. 1% of each Lysate (L), unbound proteins (F), first washing solution (W) and 10% of the Elution fractions (E) were used for SDS-PAGE controls. Samples were prepared for proteomic analysis as previously described (13 (link)).
Ip buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
IP buffer is a solution used in laboratory settings to extract and purify proteins from biological samples. It is designed to maintain the native structure and function of proteins during the immunoprecipitation (IP) process. The buffer contains components that help to solubilize and stabilize proteins, while minimizing non-specific binding interactions.
Automatically generated - may contain errors
Lab products found in correlation
Protein a g agarose, by Thermo Fisher Scientific (5 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Clean blot ip detection kit hrp, by Thermo Fisher Scientific (2 mentions)
Protein a g sepharose beads, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Phosphatase inhibitor, by Roche (2 mentions)
Protease and phosphatase inhibitor, by Merck Group (2 mentions)
Igg xp isotype magnetic beads, by Cell Signaling Technology (2 mentions)
Ha conjugated magnetic beads, by Cell Signaling Technology (2 mentions)
Protease inhibitor, by Abcam (2 mentions)
Rnase inhibitor, by Abcam (2 mentions)
Anti gp130 antibody, by Merck Group (2 mentions)
Pierce anti c myc magnetic beads, by Thermo Fisher Scientific (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
Elution buffer, by Thermo Fisher Scientific (1 mentions)
α flag magnetic beads, by Merck Group (1 mentions)
Dynabeads co immunoprecipitation kit, by Thermo Fisher Scientific (1 mentions)
Pierce crosslink magnetic ip co ip kit, by Thermo Fisher Scientific (1 mentions)
26 protocols using ip buffer
1
Immunoprecipitation of CLPP variants
2
Pan-14–3-3 Protein Immunoprecipitation
3
Co-Immunoprecipitation Assay in Rice
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For Co‐IP assays, the combinations of OsRLP1‐MYC/OsSOBIR1‐FLAG or GFP‐MYC/OsSOBIR1‐FLAG (as a negative control) were introduced into rice protoplasts for 12 h as previously described (Zhang et al., 2011 ). The Co‐IP assay was conducted as described previously (Zhang et al., 2020 (link)). Briefly, the native protein was extracted using IP buffer (50 mm Tris‐HCl, pH = 8.0, 1 mm MgCl2, 0.5 m sucrose, 10 mm EDTA with 10 mm DTT) (Thermo Scientific, Waltham, MA, USA, Cat. no. 87788) with the addition of 100 μm protease inhibitor cocktail (Roche, Basel, Switzerland, Cat. no. 04693132001) for 10 min at 4 °C and subsequently centrifuged at 12 000 g , 4 °C for 10 min (Wu et al., 2019 (link)). The supernatant was incubated with 10 μl Pierce™ anti‐c‐Myc magnetic beads (Thermo Scientific, Waltham, MA, USA, Cat. no. 88844) in 2 mL centrifuge tube for 1–2 h at 4 °C. Importantly, the beads were pre‐washed three times with 1 × PBS before using. The immunoprecipitates were then washed at least three times with 1 × PBS and then re‐suspended in 50 μL 2 × SDS sample buffer. Subsequently, the protein samples were boiled at 95–100 °C for 10 min and Western blots were performed.
4
Immunoprecipitation of Heart Tissue Proteins
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Heart tissues were lysed with immunoprecipitation (IP) buffer (Thermo) supplied with protease inhibitor cocktail (Roche) at 4 °C using a glass homogenizer to facilitate tissue lysis. Samples were then centrifuged at 13,000 g for 20 min, the lysates were precleared and 3 mg total protein/IP was incubated with the indicated primary antibodies overnight at 4 °C with rotation before incubation with Protein G Dynabeads (Invitrogen) for 6 h at 4 °C. The beads were then washed with high salt buffer once and IP buffer five times. 100 µL 3 × SDS loading buffer was added to the beads and boiled at 95 °C for 10 min prior to Western blot detection.
5
Immunoprecipitation Assay Protocol
6
Immunoprecipitation and Western Blot
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Cellular protein was collected by using the IP Buffer (Thermo Fisher Scientific, MA, USA). Protein lysates of (200 mg) were immunoprecipitated with primary antibodies or control IgG using Dynabeads Coimmunoprecipitation Kit (Thermo Fisher Scientific) and then subjected to electrophoresis on a sodium dodecyl sulphate-polyacrylamide gel. Equal amounts of protein (30 mg) were loaded on the gel and subjected to WB assay. The target proteins were probed with the primary antibodies followed by corresponding secondary antibodies.
7
Immunoprecipitation Assay Protocol
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For immunoprecipitation assays (IP), cells were lysed with cold IP buffer (87787, Thermo, Rockford, IL) supplemented immediately before use with phosphatase inhibitors (04906837001, Roche, Germany) and protease inhibitors (05892970001, Roche, Germany). The precleared samples were immunoprecipitated with primary antibody followed by protein A/G sepharose beads (sc-2003, Santa Cruz, CA) and the bound proteins were analyzed by western blot technique as described earlier 49 (link). To avoid the influence of light and heavy chain bands, IPs involved with HDAC3 and Myc were analyzed using Clean-Blot™ IP Detection Kit (HRP) (21232, Thermo, Rockford, IL).
8
HA-tagged Protein Immunoprecipitation and RNase Treatment
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2 × 106 iSLK.KSHV219 cells were transfected with eF4E2-HA, eIF4E1-HA and HIF2α-HA 24 h prior reactivation. Then, 24 h or 48 h post-reactivation, cells were washed with 1X PBS and harvested with IP buffer (ThermoFisher Scientific) containing phosphatase and protease inhibitors (Sigma). 5% of input sample was removed before IP. Then, IgG XP Isotype magnetic beads (Cell Signaling) and HA-conjugated magnetic beads (Cell Signaling) were added to the respective sample following manufacture’s recommendation. The samples containing beads were rotated at 4°C for 6 h. Then beads were washed 4 times prior to RNase treatment. The beads were treated with RNase or left untreated for 20 min at 25°C with rotation. After RNase incubation, the beads were washed 4 additional times and resuspended in 50 μL of 2X Laemmli buffer containing BME, boiled for 5 min and resolved in SDS-PAGE gel.
9
Immunoblotting and Co-immunoprecipitation Protocols
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For immunoblotting, cells were lysed either in 1% Triton X-100 or in TBS pH 7.6 with Roche complete protease inhibitor for 30 min on ice, followed by pelleting of the insoluble material via centrifugation. Lysates were heated to 100 °C in SDS sample buffer with 50 mM DTT for 10 min, separated via SDS-PAGE, and transferred to a PVDF membrane (Millipore). Membranes were blocked in 5% BSA in TBS and probed with the indicated antibodies, and reactive bands were visualized using West Pico (Thermo Fisher Scientific).
For co-immunoprecipitation experiments, cells were lysed in IP buffer (#87787, Thermo Scientific) plus Roche complete protease inhibitor for 10 min on ice, followed by the addition of Benzonase (Sigma) for 25 min at room temperature. Then, the lysate was centrifuged at 15 000 rpm and 4 °C to remove the precipitation. Then, the supernatants were incubated with the primary antibody at a slow rotating speed at 4 °C overnight, followed by addition of protein A or protein G agarose beads and incubation for a further 2 h at 4 °C. After four washes in PBST (PBS with 0.01% Tween 20), samples were eluted in SDS sample buffer with 50 mM DTT for 10 min at 100 °C, separated via SDS-PAGE and immunoblotted as described.
For co-immunoprecipitation experiments, cells were lysed in IP buffer (#87787, Thermo Scientific) plus Roche complete protease inhibitor for 10 min on ice, followed by the addition of Benzonase (Sigma) for 25 min at room temperature. Then, the lysate was centrifuged at 15 000 rpm and 4 °C to remove the precipitation. Then, the supernatants were incubated with the primary antibody at a slow rotating speed at 4 °C overnight, followed by addition of protein A or protein G agarose beads and incubation for a further 2 h at 4 °C. After four washes in PBST (PBS with 0.01% Tween 20), samples were eluted in SDS sample buffer with 50 mM DTT for 10 min at 100 °C, separated via SDS-PAGE and immunoblotted as described.
10
Investigating Gene Relationships in Osteoblasts
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An RNA immunoprecipitation (RIP) assay was performed to evaluate the relationships among genes in osteoblasts. After cells were lysed with IP buffer (Thermo Fisher Scientific), the cell lysates were centrifuged at 12,000 × g for 30 min, and the supernatant was the collected. Subsequently, 20 µl of protein magnetic beads (Millipore, Darmstadt, Germany) was added and incubated with the lysate overnight at 4 °C with anti-Ago2 antibodies and IgG. Finally, the RNAs purified from the beads using TRIzol reagent were detected using RT-qPCR.
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