Direct magnetic ip co ip kit

Manufactured by Thermo Fisher Scientific

The Direct Magnetic IP/Co-IP Kit is a laboratory tool designed for the isolation and purification of protein complexes. It utilizes magnetic beads coated with specific antibodies to capture target proteins and their interacting partners from cell or tissue samples. The kit provides a streamlined approach for performing immunoprecipitation and co-immunoprecipitation experiments, enabling researchers to study protein-protein interactions and protein complexes.

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Lab products found in correlation

Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions) Bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Pvdf membrane, by Bio-Rad (2 mentions) Ab2254, by Abcam (1 mentions) Ab3446, by Abcam (1 mentions) Ab17464, by Abcam (1 mentions) Ab2254, by Proteintech (1 mentions) Magnetic bead, by Thermo Fisher Scientific (1 mentions) Nuclear and cytoplasmic protein extraction kit, by Transgene (1 mentions) B900610, by Proteintech (1 mentions) Magnetic stand, by Thermo Fisher Scientific (1 mentions) Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions) Mouse igg1 kappa, by Thermo Fisher Scientific (1 mentions) Anti fgfr3, by Cell Signaling Technology (1 mentions) Anti akt, by Cell Signaling Technology (1 mentions) Anti p akt, by Cell Signaling Technology (1 mentions) Anti ha, by Cell Signaling Technology (1 mentions) Anti myc, by Cell Signaling Technology (1 mentions) Anti pten, by Cell Signaling Technology (1 mentions) Anti tet2, by Cell Signaling Technology (1 mentions)

6 protocols using direct magnetic ip co ip kit

Protein Extraction and Co-Immunoprecipitation

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Total protein was extracted with RIPA lysate, and cytoplasmic/nuclear proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (TransGen Biotech). After determining the concentration by the BCA method, proteins were heated at 99°C for 10 min, subjected to SDS‐PAGE gel electrophoresis, and transferred to the PDVF membrane. Immunoblots were carried out with primary antibodies, including anti‐NFAT5 (Abcam, ab3446), anti‐AURKB (Abcam, ab2254), anti‐Phospho‐Ser/Thr (Abcam, ab17464), anti‐AQP4 (Abcam, ab2254), anti‐GAPDH (ZENBIO, 20030–67E4), anti‐Histone 3 (CST, 4499s).
Co‐IP assay was performed using a Direct Magnetic IP/CO‐IP Kit (Thermo Fisher Scientific, 88 824). Briefly, rat SDH tissue was lysed with IP lysis buffer supplied with protease and phosphatase inhibitors, and then incubated with the primary antibodies: anti‐NFAT5 (Novus Biologicals, NB1203446), or anti‐AURKB (Abcam, ab2254), or anti‐IgG (Proteintech, B900610). Magnetic beads (Thermo Fisher Scientific, 88 824) were used to pull down the corresponding protein complex. After eluted the proteins from the beads, and western blot was used to identify the specific protein.

He L., Ma S., Ding Z., Huang Z., Zhang Y., Xi C., Zou K., Deng Q., Huang W.J., Guo Q, & Huang C. (2024). Inhibition of NFAT5‐Dependent Astrocyte Swelling Alleviates Neuropathic Pain. Advanced Science, 11(11), 2302916.

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Protein-Protein Interaction Assay

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P53 antibody was coupled to magnetic beads with borate buffer using the Direct Magnetic IP/Co-IP Kit (Thermo) according to the manufacturer's instructions. Next CTX1, Nutlin-3, 9-aminoacridine or DMSO (5uM final concentration) were added to individual tubes containing 100ng of recombinant HdmX. The mixture was incubated at 4°C for 90 minutes under mixing. 100ng of recombinant p53 was added to each tube and it was incubated for another 2 hours at 4°C. The antibody coupled beads were then added for 2 hours at room temperature on a mixer. The beads were washed twice with IP lysis buffer and the bound protein was eluted with sample buffer and assessed by western blot.

Karan G., Wang H., Chakrabarti A., Karan S., Liu Z., Xia Z., Gundluru M., Moreton S., Saunthararajah Y., Jackson M.W., Agarwal M.K, & Wald D.N. (2016). Identification of a small molecule that overcomes HdmX-mediated suppression of p53. Molecular cancer therapeutics, 15(4), 574-582.

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Immunoprecipitation Analysis of PFKFB3

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Immunoprecipitation analysis were performed using the Direct Magnetic IP/Co-IP Kit (Thermo Fisher, 88828) according to the manual. Briefly, PFKFB3 antibody or normal rabbit IgG (as negative control) were incubated with magnetic beads (25 μL) for 60 min. RPTCs or kidney cortex tissues were washed twice with cold PBS, and then incubated on ice with the lysis buffer from the Direct Magnetic IP/Co-IP Kit. Super-natants were collected by centrifugation. After protein measurement, 1μg/μL protein were added into the tube with antibody-coupled magnetic beads and incubated for 2 h at room temperature on a rotator. Precipitated proteins were eluted from beads and prepared for Western blot analysis. For Western blot analysis, proteins were separated by 10% SDS-PAGE and analyzed by immunoblotting using a standard method.

Wen L., Wei Q., Livingston M.J., Dong G., Li S., Hu X., Li Y., Huo Y, & Dong Z. (2022). PFKFB3 mediates tubular cell death in cisplatin nephrotoxicity by activating CDK4. Translational research : the journal of laboratory and clinical medicine, 253, 31-40.

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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.

Huang S., Xie Z., Han J., Wang H., Yang G., Li M., Zhou G., Wang Y., Li L., Li L., Zeng Z., Yu J., Chen M, & Zhang S. (2023). Protocadherin 20 maintains intestinal barrier function to protect against Crohn’s disease by targeting ATF6. Genome Biology, 24, 159.

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Immunoprecipitation and Western Blot Analysis

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Jin Z., Lu Y., Wu X., Pan T., Yu Z., Hou J., Wu A., Li J., Yang Z., Li C., Yan M., Yan C., Zhu Z., Liu B., Qiu W, & Su L. (2021). The cross-talk between tumor cells and activated fibroblasts mediated by lactate/BDNF/TrkB signaling promotes acquired resistance to anlotinib in human gastric cancer. Redox Biology, 46, 102076.

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Immunoprecipitation and Western Blot Analysis

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Immunoprecipitation analyses were performed using the Direct Magnetic IP/Co-IP Kit (ThermoFisher, 88828) according to the manual. Briefly, indicated antibodies were bound to the beads for 60 min. Cell lysates were incubated with antibody-bound beads overnight at 4 °C. Washed the beads twice with Wash Buffer and once with ultrapure water. Eluted bound antigen.
For Western blot analyses, the cells were digested in RIPA buffer with presence of Protease Inhibitor Cocktail (ThermoFisher, 87785). Protein concentration was quantified using the BCA Protein Assay Kit (ThermoFisher, 23227). After electrophoresis in SDS-PAGE, the proteins were transferred to PVDF membranes (Bio-Rad, 1620177). After blocking with PBS containing 5% nonfat milk, blots were immunoblotted with the indicated primary antibodies. The antibodies included anti-HA (Cell Signaling technology, #3724) and anti-myc (Cell Signaling technology, 2276), anti-FGFR3 (Cell Signaling technology, #4574), anti-PTEN (Cell Signaling Technology, #9599), anti-Akt (Cell Signaling Technology, #4691), anti-p-Akt (Cell Signaling Technology, #4060), anti-TET2 (Cell Signaling Technology, #18950) and anti-GAPDH (Cell Signaling technology, #5174).

Jin Z., Feng H., Liang J., Jing X., Zhao Q., Zhan L., Shen B., Cheng X., Su L, & Qiu W. (2020). FGFR3△7–9 promotes tumor progression via the phosphorylation and destabilization of ten-eleven translocation-2 in human hepatocellular carcinoma. Cell Death & Disease, 11(10), 903.

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