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Catch and release v2.0 reversible immunoprecipitation system

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Catch and Release v2.0 Reversible Immunoprecipitation System is a laboratory equipment designed for the isolation and purification of target proteins from complex biological samples. The system utilizes a reversible binding process to capture and release the proteins of interest, enabling efficient protein separation and analysis.

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Lab products found in correlation

Protease inhibitor, by Roche (3 mentions) Aj1374a, by Abcepta (2 mentions) Bis tris gel, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail tablet, by Roche (1 mentions) Bradford protein assay, by Bio-Rad (1 mentions) Quick rna microprep kit, by Zymo Research (1 mentions) Anti fmrp, by Cell Signaling Technology (1 mentions) Anti tdp 43, by Proteintech (1 mentions) Phosphatase inhibitor cocktail, by Roche (1 mentions) Mg132, by Merck Group (1 mentions) Anti gfp, by OriGene (1 mentions) Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions) Pcmv6 anxa10 gfp, by OriGene (1 mentions) Anti cul4a, by Abcam (1 mentions) Pbabe cul4a myc his, by OriGene (1 mentions) Smad6, by Abcam (1 mentions) Foxf2, by Abcam (1 mentions) Anti xbp1, by Cell Signaling Technology (1 mentions) Rabbit anti mouse igg, by Cell Signaling Technology (1 mentions) Hrp conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Catch and Release v2.0 (5 citations) Catch and Release Reversible Immunoprecipitation System (2 citations)

22 protocols using catch and release v2.0 reversible immunoprecipitation system

1

Co-immunoprecipitation of TDP-43 and FMRP

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Cells/forebrain tissue were lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS) containing a protease inhibitor cocktail tablet (Roche). Extracts were quantified with the Bradford protein assay (Bio-rad). Protein extracts (0.5 mg-1 mg) were diluted to 0.5 mg/ml with lysis buffer.
Co-immunoprecipitation was performed using the Catch and Release v2.0 Reversible Immunoprecipitation System (Millipore) according to the manufacturer's instructions with either 4 μg anti-TDP-43 (Proteintech, Cat# 10782-2 AP) or 4 μg of anti-FMRP (Cell Signaling Technology, Cat# 4317). For western blot analysis, precipitated proteins were washed and subsequently eluted in denaturation buffer and heated at 94 °C for 3 min. For RNA-immunoprecipitation analysis, precipitated proteins were washed and subsequently eluted in non-denaturation buffer, and RNAs in the immunoprecipitates were purified using Quick-RNA™ Microprep Kit (ZYMO RESEARCH) according to the manufacturer's instructions.
Wong C.E., Jin L.W., Chu Y.P., Wei W.Y., Ho P.C, & Tsai K.J. (2021). TDP-43 proteinopathy impairs mRNP granule mediated postsynaptic translation and mRNA metabolism. Theranostics, 11(1), 330-345.
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2

Immunoprecipitation and Immunoblotting Analysis

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Cells were lysed in lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4, 1 mM NaF, and 1 µg/ml aprotinin). Cell lysates containing an equal amount of protein (1.2 mg) were incubated with 4 μg anti-AR or anti-FAK antibody at 4 °C overnight. Protein–protein interactions were studied by performing immunoprecipitation using a Catch and Release v2.0 reversible immunoprecipitation system (Millipore Corporation), as per the manufacturer’s instructions. Thereafter, proteins in samples were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred to PVDF membranes, and immunoblotted with anti-AR (Millipore PG-21 #06-680), anti-ARA55 (BD Biosciences #611164) or anti-FAK antibody (BD Biosciences #610088).
Lan K.C., Wei K.T., Lin P.W., Lin C.C., Won P.L., Liu Y.F., Chen Y.J., Cheng B.H., Chu T.M., Chen J.F., Huang K.E., Chang C, & Kang H.Y. (2022). Targeted activation of androgen receptor signaling in the periosteum improves bone fracture repair. Cell Death & Disease, 13(2), 123.
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3

Investigating ANXA10-Cul4A Interaction

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Then, 293T cells were transiently co-transfected with pBabe-Cul4A-myc-his and pCMV6-ANXA10-GFP (OriGene, Rockville, MD, USA) vectors using Lipofectamine 2000 transfection reagent (Invitrogen). Twenty-four hours after transfection, the cells were treated with 10 μg/mL of MG132 (Sigma) for 24 h, and then harvested in a NP-40 lysis buffer (150 mM NaCl, 50 mM Tris [pH 8.0], 1% NP40), protease inhibitor, and phosphatase inhibitor cocktail (Roche, Lewes, UK, USA). Immunoprecipitation was performed using the Catch and Release v2.0 Reversible Immunoprecipitation System (Millipore) according to the manufacturer’s protocols. Anti-GFP (OriGene) and Anti-Cul4A (Abcam) antibodies were used for immunoprecipitation.
Hung M.S., Chen Y.C., Lin P.Y., Li Y.C., Hsu C.C., Lung J.H., You L., Xu Z., Mao J.H., Jablons D.M, & Yang C.T. (2019). Cul4A Modulates Invasion and Metastasis of Lung Cancer through Regulation of ANXA10. Cancers, 11(5), 618.
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4

Foxf2 and Smad6 Interaction in hESCs

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HESCs were harvested after stimulation of TGF‐β1 for 72 hours. Catch and Release® v2.0 Reversible Immunoprecipitation System (Millipore, USA) was used for Co‐IP test. The procedure followed the manufacturer's instruction. The cells were lysed, and then, 2 μg/mL of antibodies (Foxf2 or Smad6) was used to precipitate proteins. Anti‐rabbit immunoglobulin G monoclonal antibody (1:1000, Sigma‐Aldrich) was used as negative control in the experiments. The precipitated proteins were resolved to SDS‐PAGE and transferred to a PVDF membrane. The membranes were incubated with primary antibodies against Foxf2 or Smad6 (Abcam) overnight at 4°C, followed with secondary anti‐rabbit horseradish peroxidase‐conjugated IgG (1:1000, CST, USA), developed in ChemiDoc™ XRS+Imaging System using the chemiluminescence method (ECL, Millipore).
Chen G., Liu L., Sun J., Zeng L., Cai H, & He Y. (2020). Foxf2 and Smad6 co‐regulation of collagen 5A2 transcription is involved in the pathogenesis of intrauterine adhesion. Journal of Cellular and Molecular Medicine, 24(5), 2802-2818.
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5

Co-IP of XBP1s and p300 in Hypoxic Cells

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RAW264.7 cells were randomly assigned into normal control, hypoxia, hypoxia +0.1% DMSO (vehicle) and hypoxia +L002 (p300 inhibitor; 5 μmol/L for 24 hours) groups. The Catch and Release® v2.0 reversible immunoprecipitation system (#17‐500, Millipore, USA) was used for co‐IP. The cells were lysed, after which 2 μg/mL anti‐XBP1s (#40435) or anti‐p300 (#86377) antibodies purchased from Cell Signalling Technology were used to precipitate the proteins. Mouse anti‐rabbit IgG (#3678, Cell Signalling Technology; 1:1000) antibody was used as a negative control. The precipitated proteins were resolved by SDS‐PAGE and transferred to PVDF membranes. The membranes were incubated with primary antibodies against Ac‐K, XBP1s, p300 and GAPDH overnight at 4℃, followed by incubation with HRP‐conjugated goat‐anti‐rabbit IgG (31460, Invitrogen, USA; 1:1000), and the signals werer developed using the ECL method.
Li W., Wang Y., Zhu L., Du S., Mao J., Wang Y., Wang S., Bo Q., Tu Y, & Yi Q. (2021). The P300/XBP1s/Herpud1 axis promotes macrophage M2 polarization and the development of choroidal neovascularization. Journal of Cellular and Molecular Medicine, 25(14), 6709-6720.
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6

PGC1α-HSF1 Interaction in Apoptosis

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Lysates from differentiated WT and PGC1α-null cells were incubated with anti-HSF1 antibody (Abgent, San Diego, CA, USA, AJ1374a) and rabbit IgG (Santa Cruz, Dallas, TX, USA, sc-2027) and performed on Catch and Release v2.0 reversible immunoprecipitation system following the manufacturer's instructions (Millipore, Billerica, MA, USA). The immune complexes were eluted and subjected to SDS-PAGE. For immunoblot detection, we used anti-PGC1α (Santa Cruz, sc-13067) and anti-HSF1 (Abgent AJ1374a antibodies). The secondary goat anti-rabbit antibody was purchased from Santa Cruz (sc-2030). For western blot, proteins were extracted from cells using RIPA buffer, consisting of 20 mM Tris, 150 mM NaCl, 1% Triton X-100 and protease inhibitors (Roche) and separated in 4–20% or 10% Bis-Tris Gel (ThermoFisher, Rockville, MD, USA), transferred onto a PVDF membrane (Pierce, ThermoFisher) and incubated with primary antibodies including anti-Cleaved Caspase-3 (Cell Signaling, 9661, Danvers, MA, USA) and anti-β-actin (Sigma, A5316), overnight at 4 °C. Novex Sharp Pre-stained Protein Standard (ThermoFisher) was used as protein molecular size marker.
Xu L., Ma X., Bagattin A, & Mueller E. (2016). The transcriptional coactivator PGC1α protects against hyperthermic stress via cooperation with the heat shock factor HSF1. Cell Death & Disease, 7(2), e2102-.
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7

Immunoprecipitation of Robo1 from mmy embryos

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Because Robo1 was about threefold less abundant in mmyslm embryos than in wild-type embryos, we used 500 mmyslm embryos and 200 wildtype embryos for each immunoprecipitation experiment. Embryos that were aged for 12 to 14 hpf were homogenized in 37.5 μl of ice-cold lysis buffer [50 mM Hepes (pH 7.2), 100 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, and 1% NP-40]. The lysates were incubated on ice for 30 min and centrifuged at 15,000g for 30min at 4°C. Thirty microliters of the supernatant was used as starting material for each IP reaction using the Catch and Release v2.0 Reversible Immunoprecipitation System (#17500; Millipore) using the manufacturer’s instructions. The columns were washed three times with 1× wash buffer (Millipore) at 2000g for 20 s, and the IP was done by adding the following to the columns in the following order: 435 μl of 1×wash buffer, 30 μl of cell lysate, 25 μl of the antibody recognizing Slit-C, and 10 μl of antibody capture affinity ligand. The columns were then incubated overnight at 4°C. The flow-through was collected, and the columns were washed three times with 1× wash buffer (2000g, 20 s) before elution in 60 μl of phosphate-buffered saline–based denaturing elution buffer (2000g, 20 s). The proteins were then resolved on 4 to 12% SDS-PAGE and immunoblotted and probed with an antibody recognizing Robo1 (1:40, mouse, 13C9; DHSB).
Manavalan M.A., Jayasinghe V.R., Grewal R, & Bhat K.M. (2017). The glycosylation pathway is required for the secretion of Slit and for the maintenance of the Slit receptor Robo on axons. Science signaling, 10(484), eaam5841.
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8

Immunoprecipitation-Western Blot Assay for TTR Autoantibodies in RA

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To ascertain the significance of increase level of TTR in the diagnosis of RA, autoantibodies in the plasma of RA patients and healthy individuals were detected using immunoprecipitation-Western blotting methods. Immunoprecipitation (IP) was performed using Catch and Release v2.0 reversible immuno-precipitation system (Millipore, USA) as per manufacturer's instructions. Briefly, plasma proteins (500 μg) of individual RA patients was incubated for 2 h in mild shaking condition at RT with anti-TTR antibody separately along with 10 μl antibody capture affinity ligand buffer. The antigen-antibody complexes were eluted with 70 μl elution buffer and separated on 12% SDS–PAGE. Proteins were transferred to nitrocellulose membrane (Millipore, USA) and blocking was carried out using 5% bovine serum albumin (BSA) for 1 h at RT with gentle shaking. Plasma of RA and healthy individuals was added to the membrane as a primary antibody for overnight incubation at 4°C. After each step, the membrane was washed with PBST. The membrane was then incubated with HRP conjugated anti-human antibody for 1 h at RT with mild shaking. Enhanced chemiluminescence assay (ECL) (Thermo-Scientific, Pierce, USA) was used to develop the membrane and autoradiographed on high performance chemiluminescence film (GE Healthcare).
Sharma S., Ghosh S., Singh L.K., Sarkar A., Malhotra R., Garg O.P., Singh Y., Sharma R.S., Bhakuni D.S., Das T.K, & Biswas S. (2014). Identification of Autoantibodies against Transthyretin for the Screening and Diagnosis of Rheumatoid Arthritis. PLoS ONE, 9(4), e93905.
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9

Quantifying Cathepsins and Cystatin C in AAA

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To determine the levels of cathepsins B, D, K, L and S and cystatin C in the serum of AAA patients and the 10 healthy volunteers, immunoprecipitation was performed using the Catch and Release v2.0 Reversible Immunoprecipitation System, according to the manufacturer's instructions (Millipore Corporation, Billerica, MA, USA). The following Abs were used for detection: cathepsin B (ab30443, Abcam), cathepsin D (ab6313, Abcam), cathepsin K (ab37259, Abcam), cathepsin L (ab49984, Abcam), cathepsin S (ab18822, Abcam) and cystatin C (rabbit polyclonal to cystatin C; ab33487, Abcam). Following immunoprecipitation, the samples underwent western blot analysis as described above.
Lohoefer F., Reeps C., Lipp C., Rudelius M., Haertl F., Matevossian E., Zernecke A., Eckstein H.H, & Pelisek J. (2014). Quantitative expression and localization of cysteine and aspartic proteases in human abdominal aortic aneurysms. Experimental & Molecular Medicine, 46(5), e95-.
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10

Kinase Activity Assay for CK1α and GRK2

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Cells were lysed in co-IP buffer, and non-denatured CK1α and GRK2 proteins for kinase assay were obtained using Catch and Release® v2.0 Reversible Immunoprecipitation System (Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. In brief, 500 μg of indicated cell lysates were incubated with anti-CK1α antibody (#sc-74582, Santa Cruz Biotechnology, Inc) or anti-GRK2 antibody (#sc-13143, Santa Cruz Biotechnology, Inc) and 10 μl of antibody capture affinity ligand in a Catch and Release v2.0 spin column. After 12 h end-over-end shaking, the column was centrifuged, washed, and then eluted with non-denaturing elution buffer. The IP-CK1α and IP-GRK2 eluates were subjected to further kinase assay using CK1α1 Kinase Enzyme System and GRK5 Kinase Enzyme System (Promega, Madison, WI, USA) respectively according to the manufacturer’s instructions. Briefly, indicated eluates were incubated with ATP/substrate Mix for 60 min at room temperature, followed by ADP detection with ADP-Glo™ Kinase Assay (Promega Madison, WI, USA).
Wu X., Xiao S., Zhang M., Yang L., Zhong J., Li B., Li F., Xia X., Li X., Zhou H., Liu D., Huang N., Yang X., Xiao F, & Zhang N. (2021). A novel protein encoded by circular SMO RNA is essential for Hedgehog signaling activation and glioblastoma tumorigenicity. Genome Biology, 22, 33.
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