Normal igg

Manufactured by Merck Group

Sourced in United States, Sweden

Normal IgG is a laboratory reagent that contains a mixture of immunoglobulin G (IgG) antibodies purified from normal human serum. IgG is the most abundant type of antibody found in the human body and plays a crucial role in the immune response. This product is commonly used in various immunological and biochemical applications, such as assay development, antibody detection, and antigen-antibody interaction studies.

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Close spelling variants of this product

Normal mouse IgG (227 citations) Normal mouse IgG control (12 citations) Normal human IgG (10 citations) Normal mouse anti-IgG (10 citations) Normal goat IgG (9 citations) Normal mouse immunoglobulin G (IgG) (8 citations) Normal Rabbit IgG 12370 (8 citations) NC normal mouse IgG (7 citations) Normal rat IgG (7 citations) Normal mouse IgG antibody (7 citations)

46 protocols using normal igg

Protein Interactions in Cellular Signaling

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Lysates from the ipsilateral hemisphere were immunoprecipitated overnight at 4 °C with mouse anti-p65 (1:100, Cell Signaling Technology)/normal IgG (1:100, Sigma-Aldrich), mouse anti-p53 (1:50, Santa Cruz Biotechnology)/normal IgG (1:50, Sigma-Aldrich), and then conjugated to nProtein A Sepharose^TM 4 Fast Flow/Protein G Sepharose^TM 4 Fast Flow (GE Healthcare Bio-Science AB, Uppsala, Sweden) for 2 h at 4 °C. The complexes were washed three times with lysis buffer, denatured and subjected to Western blotting using rabbit anti-acetyl-lysine (1:1000, Cell Signaling Technology), mouse anti-p53 (1:500, Santa Cruz Biotechnology), rabbit anti-NF-κB p65 (acetyl K310) (1:3000, Abcam), and mouse anti-p65 (1:1000, Cell Signaling Technology).

Li M., Li S.C., Dou B.K., Zou Y.X., Han H.Z., Liu D.X., Ke Z.J, & Wang Z.F. (2020). Cycloastragenol upregulates SIRT1 expression, attenuates apoptosis and suppresses neuroinflammation after brain ischemia. Acta Pharmacologica Sinica, 41(8), 1025-1032.

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ChIP-qPCR Analysis of Histone Modifications

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As previously described^{17 (link)}, chromatin immunoprecipitation (ChIP) was performed using EZ-Magna ChIP A and EZ-Magna ChIP G Kits (Millipore, USA), following the manufacturer’s instructions. Briefly, ChIP grade antibodies were as follows: anti-histone H3 lysine 27 acetylation (H3K27ac; Thermo Fisher, USA), anti-histone H3 lysine 4 trimethylation (H3K4me3; Millipore, USA), anti-histone H3 lysine 27 trimethylation (H3K27me3; Millipore, USA), anti-P300 (Santa Cruz Biotechnology, USA), anti-CBP (Thermo Fisher, USA), anti-SMYD3 (Millipore, USA), and normal IgG (Millipore, USA). Immunoprecipitated DNA was amplified by qPCR, and normalized to the input DNA. Primer sequences for the promoter region of NUF2 are listed in Supplementary Table S1.

Xie X., Lin J., Fan X., Zhong Y., Chen Y., Liu K., Ren Y., Chen X., Lai D., Li X., Li Z, & Tang A. (2021). LncRNA CDKN2B-AS1 stabilized by IGF2BP3 drives the malignancy of renal clear cell carcinoma through epigenetically activating NUF2 transcription. Cell Death & Disease, 12(2), 201.

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Chromatin Immunoprecipitation of ARID2 and DNMT1

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Cells were crosslinked with 1% formaldehyde, quenched in a glycine solution, and subjected to CHIP assay using an enzymatic chromatin IP kit (#9003, CST) according to the manufacturer’s protocol. Anti-AT-rich interactive domain-containing protein 2 (ARID2) (orb386710, Biobyt), anti-DNA methyltransferase 1 (DNMT1) (61467, active motif), and normal IgG (Millipore) antibodies were used for immunoprecipitation. ChIP-enriched DNA samples were quantified by qPCR to determine ARID2- and DNMT1-binding sites in the IFN-γ promoter region. Data were shown as relative enrichment following normalisation to control IgG. The primer sequences used for ChIP-qPCR are shown in Supplementary Table S1.

Zhou C., Zhang Y., Yan R., Huang L., Mellor A.L., Yang Y., Chen X., Wei W., Wu X., Yu L., Liang L., Zhang D., Wu S, & Wang W. (2020). Exosome-derived miR-142-5p remodels lymphatic vessels and induces IDO to promote immune privilege in the tumour microenvironment. Cell Death and Differentiation, 28(2), 715-729.

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RNA Immunoprecipitation of Ago2 Protein

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The Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, U.S.A.) was applied to conduct RNA immunoprecipitation (RIP) assay. U251 and LN229 cells (1 × 10⁷) were reaped and rinsed in ice-cold PBS. Thereafter, cells were lysed in the RIP lysis buffer at 4°C for 30 min. Cell lysates (100 μl) were cultured with magnetic beads conjugated with human antibodies against argonaute 2 (Ago2) (Millipore, U.S.A.) or normal IgG (Millipore, U.S.A.). Proteinase K buffer was applied to digest proteins. The immunoprecipitated RNA and total RNA were extracted from the whole cell lysates (input control) and analyzed by qRT-PCR examination. Each experimental procedure was repeated more than three times.

Liu X., Chen R, & Liu L. (2019). SP1–DLEU1–miR-4429 feedback loop promotes cell proliferative and anti-apoptotic abilities in human glioblastoma. Bioscience Reports, 39(12), BSR20190994.

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ChIP-qPCR Analysis of Grem-1 Promoter

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Kidneys were fixed in 1% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) followed by quenching with 0.125M glycine. DNA fragments 500–1000 bp long were generated on a BioRuptor (Diagenode), and ChIP assays were performed using the High Cell ChIP kit (Diagenode, Denville, NJ, USA) following the manufacturer’s instructions. The antibodies used were BRD4 (Bethyl Laboratories, Burlington, MA, USA) and normal IgG as negative control (Millipore). Immunoprecipitated DNA was analyzed by quantitative RT-PCR using the following primers of Grem-1 promoter:

Forward 5′-GACCAATGGAGAGACGCAGT-3′

Reverse 5′-GTTCTTCGCTGTGGACGAGT-3′

Chromatin obtained before immunoprecipitation was used as the input control. Relative enrichment was calculated as the percentage of input DNA for each sample using the formula % input = 2 exp [(Ct unbound) − log₂ (unbound dilution factor) – Ct bound)] × 100 and normalized to normal rabbit IgG antibody (considered as 1).

Tejedor-Santamaria L., Morgado-Pascual J.L., Marquez-Exposito L., Suarez-Alvarez B., Rodrigues-Diez R.R., Tejera-Muñoz A., Marchant V., Mezzano S., Lopez-Larrea C., Sola A., Fernandez-Juarez G.M., Ortiz A., Rayego-Mateos S, & Ruiz-Ortega M. (2022). Epigenetic Modulation of Gremlin-1/NOTCH Pathway in Experimental Crescentic Immune-Mediated Glomerulonephritis. Pharmaceuticals, 15(2), 121.

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HMBOX1 ChIP-qPCR protocol

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Cells were cross linked with 1% formaldehyde and quenched in glycine solution and ChIP assays performed using an Enzymatic ChIP Kit (#9003; CST, Danvers, MA, USA), according to the manufacturer’s protocol. Anti-HMBOX1 antibodies (16123-1-AP; Proteintech, Chicago, IL, USA) and normal IgG (Millipore, Burlington, MA, USA) were used for immunoprecipitation, whereas ChIP-enriched DNA samples were quantified by qPCR to determine HBSs in the SOCS1 promoter region. Data were shown as relative enrichment normalized to control IgG. The primer sequences used for ChIP-qPCR are presented in Table S1.

Zhou C., Wei W., Ma J., Yang Y., Liang L., Zhang Y., Wang Z., Chen X., Huang L., Wang W, & Wu S. (2021). Cancer-secreted exosomal miR-1468-5p promotes tumor immune escape via the immunosuppressive reprogramming of lymphatic vessels. Molecular Therapy, 29(4), 1512-1528.

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RIP Assay for RNA Enrichment

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The RIP kit (BersinBio, Guangzhou, China) was used according to recommended procedures. BMSCs were collected and lysed in radioimmunoprecipitation (RIPA) lysis buffer. Magnetic beads were then incubated with 10 μg antibodies and normal IgG (Millipore, MA, USA) overnight at 4 °C. Immunoprecipitated RNAs were extracted using TRIzol, and RNA enrichment was analyzed using qRT-PCR.

You Y., Liu J., Zhang L., Li X., Sun Z., Dai Z., Ma J., Jiao G, & Chen Y. (2023). WTAP-mediated m6A modification modulates bone marrow mesenchymal stem cells differentiation potential and osteoporosis. Cell Death & Disease, 14(1), 33.

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Immunoprecipitation and Western Blot Analysis

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Cells were washed twice with ice-cold PBS and harvested in cold sonication buffer [150 mM NaCl, 50 mM Tris (pH 8), 1% NP-40, 0.5% deoxycholate, 25 mM MG132, and 2X protease inhibitor mixture (Roche, cOmplete EDTA-free)]. Cells were sonicated (QSonica Sonicator system fitted with a 1.6-mm tip) and centrifuged at 9391 RCF for 5 min at 4°C. Lysates were incubated overnight at 4°C with specific antibodies or normal IgG (Millipore, Billerica, MA). Lysates were then incubated with protein A/G beads (Santa Cruz Biotechnology) for 3 h, followed by 4 washes with ice cold wash buffer [50 mM NaCl, 20 mM Tris (pH 8.3), 0.5% Sodium deoxycholate, 0.5% Nonidet P-40, 2 mM EDTA, 25 μM MG132, and 2X protease inhibitor mixture]. Immunoprecipitated proteins were resolved by SDS/PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore). Blots were incubated with different primary antibodies followed by incubation with HRP-conjugated secondary antibodies. For CBFβ IP, Clean-Blot IP Detection Reagent was used (Cat. #21230, Thermo Scientific, Rockford, IL). Chemiluminescence (Perkin-Elmer Life Sciences, Boston, MA) was visualized using the BioRad ChemiDoc SRS device.

Lopez-Camacho C., van Wijnen A.J., Lian J.B., Stein J.L, & Stein G.S. (2014). Core Binding Factor β (CBFβ) and the Leukemogenic Fusion Protein CBFβ-Smooth Muscle Myosin Heavy Chain (SMMHC) Associate with Mitotic Chromosomes to Epigenetically Regulate Ribosomal Gene Expression. Journal of cellular biochemistry, 115(12), 2155-2164.

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RIP Assay of LINC01426-miR-519d-5p Interaction

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RIP assay was performed to detect the binding between LINC01426 and miR-519d-5p using a Magna RIP RNA-Binding Protein Immunoprecipitation kit (EMD Millipore). NSCLC cells were lysed in RIP buffer, and the cell extracts were incubated with magnetic beads conjugated with anti-Ago2 antibody (Millipore) or normal IgG (Millipore). Prior to immunoprecipitated RNA extraction, the magnetic beads were harvested after overnight incubation at 4°C, rinsed with RIP wash buffer, and treated with proteinase K to remove protein. The immunoprecipitated RNA was measured using qRT-PCR.

Dai J., Wang B., Zhao Y., Zuo X., Cui H., Chen X, & Liu X. (2020). Long Noncoding RNA LINC01426 Sequesters microRNA-519d-5p to Promote Non-Small Cell Lung Cancer Progression by Increasing ETS1 Expression. Cancer Management and Research, 12, 12697-12708.

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Ago2-Mediated RNA Immunoprecipitation

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An EZ-Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore,) was used to conduct the RIP assay. Cells were collected and lysed in pre-cooled lysis buffer supplemented with 1 mM PMSF, protease inhibitor and RNase inhibitor. Cells were then incubated with RIP buffer containing magnetic beads conjugated to a human anti-Argonaute 2 (Ago2) antibody (Millipore) or normal IgG (Millipore). The precipitate was digested, and the co-immunoprecipitated RNA was then isolated for PCR.

Chen F., Xu B., Li J., Yang X., Gu J., Yao X, & Sun X. (2021). Hypoxic tumour cell-derived exosomal miR-340-5p promotes radioresistance of oesophageal squamous cell carcinoma via KLF10. Journal of Experimental & Clinical Cancer Research : CR, 40, 38.

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