Anti o glcnac rl2

Manufactured by Abcam

Anti-O-GlcNAc (RL2) is a mouse monoclonal antibody that recognizes the O-linked N-acetylglucosamine (O-GlcNAc) modification on proteins. It can be used to detect and study O-GlcNAcylated proteins.

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9 protocols using anti o glcnac rl2

Profiling Protein Acetylation and O-GlcNAcylation

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Cells and tumors were lysed in a 1% Nonidet P-40, 50 mM Tris-HCl, 150 mM NaCl, 0.1 mM EDTA buffer with protease, protein phosphatase, and O-GlcNAcase inhibitors. Proteins were immunoprecipitated by incubating whole cell lysates/tumor homogenates with the indicated antibodies for 2 hours and precipitating with Protein A/G agarose beads. Whole cell lysates/tumor homogenates and immunoprecipitation samples were subjected to 8% SDS-PAGE gel electrophoresis and transferred to PVDF membranes. Membranes were incubated with the indicated primary antibodies overnight at 4°C. Western blots were visualized using HRP-conjugated secondary antibodies and ECL. The following antibodies were used: anti-actin (Sigma), anti-acetyl-lysine (AcK, Cell Signaling), anti-Flag (Sigma), anti-HN (Clontech), anti-OGT (Cell Signaling), anti-OGA (Novus), anti-O-GlcNAc (RL2, Abcam), and anti-PKM2 (Cell Signaling).

Singh J.P., Qian K., Lee J.S., Zhou J., Han X., Zhang B., Ong Q., Ni W., Jiang M., Ruan H.B., Li M.D., Zhang K., Ding Z., Lee P., Singh K., Wu J., Herzog R.I., Kaech S., Wendel H.G., Yates JR I.I.I., Han W., Sherwin R.S., Nie Y, & Yang X. (2019). O-GlcNAcase targets pyruvate kinase M2 to regulate tumor growth. Oncogene, 39(3), 560-573.

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Immunoblot Analysis of O-GlcNAc Modification

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IP and immunobloting experiments were performed as previously described (42 (link)). O-GlcNAc was enriched by treating the cells with Glu plus TMG as previously described (treated with 5 μM TMG for 24 h and 30 mM Glu for 3 h) (41 (link)). Antibodies used were as follows: anti-ULK1 (CST; catalog no.: 8054), anti-OGT (Santa Cruz; catalog no.: sc-32921), anti-O-GlcNAc (RL2) (Abcam; catalog no.: ab2739), anti-O-GlcNAc (CTD110.6) (Sigma; catalog no.: O7764), LC3B (CST; catalog no.: 2775S), and anti-STX17 (Merck; catalog no.: HPA001204).
Peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch. Blotted proteins were visualized using the ECL detection system (Amersham). Signals were detected by a LAS-4000 (Fujifilm) and quantitatively analyzed by densitometry using the Multi Gauge software (Fujifilm). All Western blots were repeated at least three times. Silver staining analysis was performed as described (41 (link)).

Shi Y., Yan S., Shao G.C., Wang J., Jian Y.P., Liu B., Yuan Y., Qin K., Nai S., Huang X., Wang Y., Chen Z., Chen X., Dong M.Q., Geng Y., Xu Z.X, & Li J. (2022). O-GlcNAcylation stabilizes the autophagy-initiating kinase ULK1 by inhibiting chaperone-mediated autophagy upon HPV infection. The Journal of Biological Chemistry, 298(9), 102341.

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Profiling Protein Acetylation and O-GlcNAcylation

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Antibody Immunochemistry Protocol

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Anti‐O‐GlcNAc (RL2, ab2739) was from Abcam. Anti‐ZO‐1 (617300) was from Life technologies. Anti‐ZO‐2 (18900‐1‐AP), anti‐CLAUDIN 3 (16456), and anti‐OCCLUDIN (13409) were from proteintech. Anti‐Ki67 (550609) was from BD Biosciences. Anti‐Myeloperoxidase (MPO, PA5‐16672) was from Thermo Fisher. Anti‐F4/80 (123102) and Anti‐CD4 (100506) were from Biolegend. Anti‐OGT (#24083), anti‐Cleaved CASPASE 3 (#9661), anti‐LC3B (#2775), anti‐α‐E‐CATENIN (#3042), anti‐β‐CATENIN (#8480), anti‐PLAKOGLOBIN (#2309), anti‐STAT1 (#9172), anti‐STAT3 (#4904), anti‐phospho‐STAT3 (Tyr705, #9145), anti‐STAT5 (#9363), anti‐NF‐κB p65 (#8242), and anti‐phospho‐NF‐κB p65 (Ser536, #3033) were from Cell Signaling Technology. Anti‐lysozyme (CSB‐PA02769A0Rb) was from Cusabio. Anti‐DESMOPLAKIN I/II (sc‐33555) was from Santa Cruz Biotechnology. Anti‐β‐ACTIN (A5441) and anti‐CLAUDIN 5 (ABT45) were from Millipore Sigma.

Zhao M., Xiong X., Ren K., Xu B., Cheng M., Sahu C., Wu K., Nie Y., Huang Z., Blumberg R.S., Han X, & Ruan H. (2018). Deficiency in intestinal epithelial O‐GlcNAcylation predisposes to gut inflammation. EMBO Molecular Medicine, 10(8), e8736.

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Western Blot Analysis of Protein O-GlcNAcylation

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The protein sample (15 μL) was loaded on 6–12% Tris–glycine SDS-PAGE gels and run on a Mini-PROTEAN® BioRad gel system. Gels were transferred with the Invitrogen iBlot. Membranes were stained with Ponceau stain to verify transfer and equal protein loading and blocked with 3% BSA + 1 × TBST for 1 h at 24 °C. Primary antibodies and the following dilutions were incubated with the membranes for 12 h: anti-HA (1:1000; Cell Signaling; 3724S), anti-O-GlcNAc RL2 (1:1000; Abcam; ab2739), and anti-GAPDH (1:1000; Cell Signaling; D4C6R). The membranes were washed 3 × 5 min each wash with 1 × TBST and incubated with the following secondary antibodies and dilutions: anti-rabbit HRP (1:10,000; Rockland; 611–1302) and anti-mouse IR 800 (1:10,000; LI-COR; 925–32,210). The membranes were washed 3 × 5 min each wash with 1 × TBST, and results were obtained by chemiluminescence or IR imaging using Azure c600. The membranes were quantified using LI-COR Image Studio Lite, and graphs were made with GraphPad Prism 9.

Ramirez D.H., Yang B., D’Souza A.K., Shen D, & Woo C.M. (2021). Truncation of the TPR domain of OGT alters substrate and glycosite selection. Analytical and bioanalytical chemistry, 413(30), 7385-7399.

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Western Blot Analysis of Aortic Proteins

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Aortic tissue lysates were prepared in SDS lysis buffer, as described earlier56 (link) and protein content was determined using BCA protein assay. Equal amounts of proteins (35 μg) were resolved on 8% SDS-PAGE and transferred to PVDF membranes. Immunoblotting was performed using anti-TSP-1 (1:500-1:1000, Neomarkers, Freemont, CA), anti-O-GlcNAc (RL2, 1:1000; Abcam, Cambridge, MA), anti-OGT (1:1000, Cell Signaling, Danvers MA), anti-PCNA (1:300, Abcam, Cambridge, MA), anti-SM-MHC (1:2000, Proteintech, Rosemont, IL) and anti-vimentin (1:1000, Cell Signaling, Danvers, MA) antibodies. Membranes were stripped and re-probed with anti-β-actin used as a loading control; equal protein loading of samples was also confirmed by staining the membranes with Ponceau S. All immunoblot images were captured using Protein Simple and densitometric analyses was performed using the Image J software.

Ganguly R., Sahu S., Ohanyan V., Haney R., Chavez R.J., Shah S., Yalamanchili S, & Raman P. (2017). Oral chromium picolinate impedes hyperglycemia-induced atherosclerosis and inhibits proatherogenic protein TSP-1 expression in STZ-induced type 1 diabetic ApoE−/− mice. Scientific Reports, 7, 45279.

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Cloning and Modification of C. elegans SKN-1 and OGT-1

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The cDNAs for skn-1, skn-1(321–623aa) and ogt-1 of C. elegans were amplified by PCR and cloned into pGEX-6P-1 and PET-28a vectors, respectively. All of the point mutations of SKN-1 were generated by site-directed mutagenesis according to the QuikChange (Stratagene) protocal. The antibodies of anti-GFP, anti-GST (Tianjin Sungene Biotech), anti-β-actin (Sigma), anti-O-GlcNAc RL2 (Abcam), anti-OGT (sigma) and anti- phosphoserine (Millipore) were purchased from the commercial channels. The anti-OGT and anti-GFP antibodies were first analyzed and make sure of their specific binding to the OGT-1 and GFP in C. elegans (Supplementary Fig. S10a,b). Rabbit polyclonal antibodies against phospho-SKN-1(Ser483) were generated by using synthetic peptides, TTDSSSTCS(#)RLSSESPRYTSE. # means the phosphorylated residue Ser483.

Li H., Liu X., Wang D., Su L., Zhao T., Li Z., Lin C., Zhang Y., Huang B., Lu J, & Li X. (2017). O-GlcNAcylation of SKN-1 modulates the lifespan and oxidative stress resistance in Caenorhabditis elegans. Scientific Reports, 7, 43601.

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Quantifying Protein Modifications by Western Blot

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For Western blot analysis, protein samples were separated by SDS/PAGE and transferred on to nitrocellulose membrane (Whatman). The membranes were blocked for 1 h in 5% non-fat milk powder or 5% BSA in TBS-0.2% Tween20 (TBS-T) buffer for 1 h at room temperature with gentle shaking. Then, the following primary antibodies, diluted in blocking buffer, were added to the membranes overnight at 4°C: anti-OGT (DM-17, Sigma–Aldrich), anti-OGT (Abcam ab177941), anti-O-GlcNAc (RL2, Abcam), anti-O-GlcNAc [C-terminal domain (CTD) 110.6, Cell Signaling], anti-TOM20 (F-10, Santa Cruz Biotechnology), anti-ATP synthase β subunit (ATPB, 3D5, Abcam), anti-Timm13 (A01, Abnova), anti-HSP60 (D307, Cell Signaling), anti-α-tubulin (12G10, Developmental Studies Hybridoma Bank, University of Iowa) and anti-α-tubulin (11H10, Cell Signaling). The following fluorescent secondary antibodies, diluted in 1% milk in TBS-T, were added to the membranes for 1 h at room temperature: donkey anti-rabbit 800, donkey anti-mouse 680, goat anti-rabbit 800 and goat anti-mouse 680 (LI-COR). Images were acquired and analysed using the LI-COR Odyssey Image System.

Trapannone R., Mariappa D., Ferenbach A.T, & vanAalten D.M. (2016). Nucleocytoplasmic human O-GlcNAc transferase is sufficient for O-GlcNAcylation of mitochondrial proteins. Biochemical Journal, 473(12), 1693-1702.

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Antibody Sourcing for Biochemical Assays

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Antibodies were purchased as follows: anti-GST 1-109 (sc-33613), anti-OGT (sc-32921), anti-CK2α (sc-12738), and anti-Nup62 (sc-48373) from Santa Cruz Biotechnology; anti-O-GlcNAc RL2 from Abcam; anti-T7 and anti-S tags from Novagen.; anti-Flag (F1804), anti-Actin, anti-O-GlcNAc (CTD110.6), and anti-His (H1029) from Sigma-Aldrich; and anti-HA (3F10) from Roche Applied Science.

Kapuria V., Röhrig U.F., Bhuiyan T., Borodkin V.S., van Aalten D.M., Zoete V, & Herr W. (2016). Proteolysis of HCF-1 by Ser/Thr glycosylation-incompetent O-GlcNAc transferase:UDP-GlcNAc complexes. Genes & Development, 30(8), 960-972.

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