Irdye 680lt goat anti rabbit igg

Manufactured by LI COR

Sourced in United States, Germany

IRDye 680LT Goat-Anti-Rabbit IgG is a secondary antibody conjugated with the IRDye 680LT fluorescent dye. It is designed for detection of rabbit primary antibodies in various immunoassay applications.

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Lab products found in correlation

Irdye 800cw goat anti mouse igg, by LI COR (23 mentions) Odyssey infrared imaging system, by LI COR (12 mentions) β actin, by Merck Group (5 mentions) α tubulin, by Merck Group (5 mentions) Image studio lite, by LI COR (4 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions) Irdye 800cw goat anti rabbit igg, by LI COR (3 mentions) E cadherin, by Cell Signaling Technology (3 mentions) Te42 tank blotting units, by Hoefer (3 mentions) Gapdh, by Abcam (2 mentions) Lamin b1, by Cell Signaling Technology (2 mentions) Ck5 mouse ncl l ck5, by Leica Biosystems (2 mentions) Transfection reagent, by Thermo Fisher Scientific (2 mentions) Progesterone, by Merck Group (2 mentions) On target pooled sirnas, by Thermo Fisher Scientific (2 mentions) Irdye 800cw goat anti mouse igg 926 32210, by LI COR (2 mentions) Alexa fluor 488 goat anti mouse a 11029, by Thermo Fisher Scientific (2 mentions) Goat anti rabbit a11008, by Thermo Fisher Scientific (2 mentions) Alexa fluor 594 goat anti mouse a11032, by Thermo Fisher Scientific (2 mentions) Goat anti rabbit a11037, by Thermo Fisher Scientific (2 mentions)

36 protocols using irdye 680lt goat anti rabbit igg

Protein Extraction and Western Blot Analysis

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The total protein was extracted from cells with ice-cold lysis buffer supplemented with protease inhibitor cocktail (Merck, Germany) and PMSF. The nuclear protein was extracted with Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China). The protein concentration was determined with Detergent Compatible Bradford Protein Assay Kit (Beyotime, Shanghai, China). Proteins were separated on SDS-PAGE gels and transferred onto nitrocellulose membrane (Life Technologies, USA). After blocking with Odyssey® blocking buffer (PBS) (LI-COR, Lincoln, USA), the membranes were incubated overnight with the primary antibodies of CD31 (ab28364,1:500), vWF (ab6994,1:500), α-SMA (ab32575,1:1000), vimentin (ab92547,1:1000), Histone H3 (ab1791,1:1000), β-catenin (ab32572,1:5000) and GAPDH (ab181602,1:10000) (abcam, Cambridge, UK). After that, IRDye 800CW Goat anti-Rabbit IgG (H + L) Secondary Antibody (926–32,211, 1:10000), IRDye® 680LT Goat anti-Rabbit IgG (H + L) Secondary Antibody (926–68,021) (LI-COR, Lincoln, USA) were added and incubated for 1 h at room temperature. Then the band intensities were scanned by Odyssey (LI-COR, Lincoln, USA) and normalized to GAPDH or Histone H3.

Li H., Zhao Q., Chang L., Wei C., Bei H., Yin Y., Chen M., Wang H., Liang J, & Wu Y. (2019). LncRNA MALAT1 modulates ox-LDL induced EndMT through the Wnt/β-catenin signaling pathway. Lipids in Health and Disease, 18, 62.

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Antibodies and Compounds for AMPK Signaling

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Antibodies for adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK/Ampk) (#2793), Flag-Tag (#2368), His-Tag (#12698), GST-Tag (#2624), GAPDH (#5174) and phosphospecific antibodies for AMPK/Ampk -pThr172 (#2535) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies for HMGCR/Hmgcr (#ab174830), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) (#ab188321), HA-Tag (#ab1424) and phosphospecific antibodies for acetyl-CoA carboxylase (ACC/Acc) pSer79 (#ab68191) were purchased from Abcam (Cambridge, UK). Antibodies for ACC/Acc (#21923-1-AP), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) (#17643-1-AP) and sterol regulatory element-binding protein 2 SREBP2/Srebp2 (#A13049) were from Proteintech (Chicago, IL, USA). Antibody for GAPDH (#GTX100118) was purchased from GeneTex (Alton, CA, USA). IRDye 800CW goat anti-Mouse IgG (#926-32213) and IRDye 680LT goat anti-Rabbit IgG (#926-68021) were purchased from LI-COR Biosciences (Lincoln, Nebraska, USA). Cholesterol (#C8667) and methyl-β-cyclodextrin (#C4555) were purchased from Sigma-ldrich (St. Louis, MO, USA). Aminoimidazole carboxamide ribonucleotide (AICAR) (#S1802) and Compound C (#S7840) were purchased from Selleckchem (Houston, TX, USA).

Wang H., Lin C., Yao J., Shi H., Zhang C., Wei Q., Lu Y., Chen Z., Xing G, & Cao X. (2019). Deletion of OSBPL2 in auditory cells increases cholesterol biosynthesis and drives reactive oxygen species production by inhibiting AMPK activity. Cell Death & Disease, 10(9), 627.

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Immunoblotting Analysis of Cell Lysates

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All cell lysates for immunoblotting were harvested in RIPA buffer. Primary antibodies were as follows: CK5 (mouse NCL-L-CK5, Leica Biosystems, Buffalo Grove, IL, USA, 1:1500), α-tubulin (ST1568, Sigma, 1:1000), β-catenin (mouse 2698, or rabbit 9587, Cell Signaling Technologies, Danvers, MA, USA, 1:1000), E-cadherin (14472, Cell Signaling, 1:1000), Lamin B1 (12586, Cell Signaling, 1:1000), CK17 (NBP2–29421, Novus Biologicals, 1:1000), or β-actin (A5441, Sigma, 1:1000). Secondary antibodies were IRDye800CW Goat-Anti-Mouse IgG (926–32210, Li-Cor Biosciences, Lincoln, NE, USA) and IRDye 680LT Goat-Anti-Rabbit IgG (926–68021, Li-Cor Biosciences) both at 1:10,000. Immunoblots were imaged and analyzed with the Odyssey Infrared Imaging System and Image Studio Lite (Li-Cor Biosciences).

McGinn O., Ward A.V., Fettig L.M., Riley D., Ivie J., Paul K.V., Kabos P., Finlay-Schultz J, & Sartorius C.A. (2020). Cytokeratin 5 alters β-catenin dynamics in breast cancer cells. Oncogene, 39(12), 2478-2492.

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Quantitative Analysis of PR and ERα

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For IP, 100–200 mg of flash frozen tumor was pulverized and homogenized in lysis buffer (50mM Tris pH 7.4, 140 nM NaCL, 2mM EGTA, 1.0% Tween-20). IP was performed overnight by combining 750 ug precleared lysate with 5 ug of antibody prebound to protein G Dynabeads (Thermo Fisher). Antibodies for IP were to PR (Santa Cruz Biotechnology, sc-7208, sc-166169; DAKO, 1294), and ERα (sc-543). Each IP was coupled with a corresponding IgG negative control of the same species. Beads were washed with wash buffer (50mM Tris pH 7.4, 140 nM NaCL, 2mM EGTA, 0.1% Tween 20), then boiled at 100°C for 10 min in 30 uL of 1× SDS loading buffer.
Antibodies for immunoblots were PR (sc-7208; DAKO, 1294), ERα (sc-56836), POLR3A (Abcam, 12825), and POLR3B (sc-515362). For imaging, the Odyssey Infrared Imaging System (Li-Cor Biosciences) was used, with secondary antibodies IRDye800CW Goat-Anti-Mouse-IgG and IRDye680LT Goat-Anti-Rabbit-IgG (Li-Cor Biosciences).

Finlay-Schultz J., Gillen A.E., Brechbuhl H.M., Ivie J.J., Matthews S.B., Jacobsen B.M., Bentley D.L., Kabos P, & Sartorius C.A. (2017). Breast cancer suppression by progesterone receptors is mediated by their modulation of estrogen receptors and RNA polymerase III. Cancer research, 77(18), 4934-4946.

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Optimized Protein Expression Techniques

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On-Target pooled siRNAs, miRNA mimics/inhibitors, and transfection reagents were obtained from Thermo-Fisher Scientific (Pittsburgh, PA, USA); pmiR-GLO luciferase vector and Dual Luciferase Reporter assay were obtained from Promega (Madison, WI, USA). shRNA vectors were from the University of Colorado Functional Genomics Facility (Boulder, CO, USA). Primary antibodies used included: Stat5 (SC-835, SC-836; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), CK5 (mouse NCL-L-CK5 and rabbit 2290-1; Vector Laboratories/Epitomics, Burlingame, CA, USA), PR (Dako, Carpinteria, CA, USA), α-tubulin and β-actin (Sigma-Aldrich, St. Louis, MO, USA). For western blot, secondary antibodies included: IRDye 800CW Goat-Anti-Mouse IgG (926-32210) and IRDye 680LT Goat-Anti-Rabbit IgG (926-68021)(Li-Cor Biosciences, Lincoln, NE, USA). For Western blot imaging, the Odyssey Infrared Imaging System (Li-Cor Biosciences). For immunocytochemistry, secondary antibodies: AlexaFluor488 goat anti-mouse (A11029) and goat anti-rabbit (A11008), AlexaFluor594 goat anti-mouse (A11032) and goat anti-rabbit (A11037) (Invitrogen/Life Technologies, Grand Island, NY, USA). Hormones (17β-estradiol, progesterone, and MPA) were purchased from Sigma. Pimozide (573110) was purchased from EMD Millipore (Billerica, MA, USA).

Finlay-Schultz J., Cittelly D.M., Hendricks P., Patel P., Kabos P., Jacobsen B.M., Richer J.K, & Sartorius C.A. (2014). Progesterone downregulation of miR-141 contributes to expansion of stem-like breast cancer cells through maintenance of progesterone receptor and Stat5a. Oncogene, 34(28), 3676-3687.

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Antibody Characterization for Viral Protein Analysis

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The following antibodies were used in the study: anti-NS5A monoclonal antibody (9E10, a kind gift from Charles M. Rice); anti-Core antigen monoclonal antibody (C7-50, Thermo Scientific); 6xHis monoclonal antibody (Clontech); monoclonal anti-Flag M2 antibody (Sigma); GST•Tag monoclonal antibody (Novagen); β-Actin, rabbit monoclonal antibody (LI-COR Biosciences); anti-TIP47, rabbit polyclonal antibody (M6PRBP1, Proteintech); anti-Calnexin, rabbit IgG fraction of antiserum (Sigma); human anti-E1 MAb 1C4 (Innogenetics); anti-E2 antibody D3.7 (DAO5, a kind gift from Arvind Patel); goat anti-ApoE polyclonal antibody (Millipore); IRDye 800CW conjugated goat anti-mouse IgG, IRDye 680LT goat anti-rabbit IgG, IRDye 680RD donkey anti-goat IgG, IRDye 680RD goat anti-human IgG (all IRDye secondary antibodies were from LI-COR Biosciences); ECL anti-mouse IgG, horseradish peroxidase linked whole antibody from sheep (GE Healthcare). All restriction enzymes were obtained from New England Biolabs. AccuPrime Pfx DNA polymerase was from Invitrogen whereas T4 DNA ligase was from Roche Applied Science. All other reagents were purchased from Sigma, Fisher, or VWR unless indicated otherwise.

Gawlik K., Baugh J., Chatterji U., Lim P.J., Bobardt M.D, & Gallay P.A. (2014). HCV Core Residues Critical for Infectivity Are Also Involved in Core-NS5A Complex Formation. PLoS ONE, 9(2), e88866.

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3D Cell Culture Lysis and Immunoblotting

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Cells were lysed in RIPA buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton-X 100, 0.5 sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 10 mM sodium fluoride and 20 mM β-glycerophosphate plus Complete protease inhibitors (Roche, Basel, Switzerland). For lysates from 3D cultures, cells were cultured on pure matrigel without collagen. Spheroids were isolated after 4 days by dissolving the matrigel in ice cold Cell Recovery Solution (BD). After centrifugation the spheroids were lysed in RIPA buffer. Lysates were clarified by centrifugation. Equal amounts of protein were separated by SDS–PAGE (NuPAGE^® Novex Bis-Tris Gel; Invitrogen) and transferred to nitrocellulose membrane (iBlot^®Gel Transfer Stacks; Invitrogen). Alternatively lysates were loaded on 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Roth). Membranes were blocked with 0.5% blocking reagent (Roche) in PBS containing 0.1% Tween-20 and incubated with primary antibodies, followed by HRP-conjugated secondary antibodies. Visualization was done with ECL detection system (Pierce, Rockford, IL, USA). Alternatively detection was done with IR-labled secondary antibodies IRDye 800 CW goat anti-mouse IgG (Licor Biotechnology, Bad-Homburg, Germany) and IRDye 680 LT goat anti-rabbit IgG (Licor Biotechnology).

Möller Y., Morkel M., Schmid J., Beyes S., Hendrick J., Strotbek M., Riemer P., Schmid S., Schmitt L.C., Kontermann R., Mürdter T., Schwab M., Sers C, & Olayioye M.A. (2016). Oncogenic Ras triggers hyperproliferation and impairs polarized colonic morphogenesis by autocrine ErbB3 signaling. Oncotarget, 7(33), 53526-53539.

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Western Blot Analysis of Androgen Receptor Signaling

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Cells were lysed on ice with RIPA lysis buffer (Thermo Fisher Scientific, #89901) supplemented with protease inhibitors (Roche, #11836170001) and quantitated using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, #23225). Equal amounts of protein were loaded onto NuPAGE 4–12% Bis-Tris Protein Gels (Thermo Fisher Scientific, #NP0335) for separation by SDS-PAGE. Proteins were transferred to nitrocellulose membranes using an iBlot2 (Thermo Fisher Scientific), followed by overnight incubation at 4°C with the following primary antibodies and dilutions. AR: Cell Signaling (#5153), 1:2,000. AR-V7: RevMAb (#31–1109-00), 1:500. PRMT1: Cell Signaling (#2449), 1:1,000. ADMA: Cell Signaling (#13522), 1:250. β-Actin: Cell Signaling (#3700), 1:5,000. Membranes were washed in TBS-T before incubation for 1 hour at room temperature with the following secondary antibodies: IRDye 680LT Goat anti-Rabbit IgG: LI-COR (#926–68021), 1:20,000. IRDye 800CW Donkey anti-Mouse IgG: LI-COR (#926–32212), 1:20,000. Immunoblots were imaged with the Odyssey CLx Infrared Imaging System (LI-COR Biosciences). Band intensity was quantitated using ImageStudioLite v5.2.5 or ImageJ 1.52v.

Tang S., Sethunath V., Metaferia N.Y., Nogueira M.F., Gallant D.S., Garner E.R., Lairson L.A., Penney C.M., Li J., Gelbard M.K., Abou Alaiwi S., Seo J.H., Hwang J.H., Strathdee C.A., Baca S.C., AbuHammad S., Zhang X., Doench J.G., Hahn W.C., Takeda D.Y., Freedman M.L., Choi P.S, & Viswanathan S.R. (2022). A genome-scale CRISPR screen reveals PRMT1 as a critical regulator of androgen receptor signaling in prostate cancer. Cell reports, 38(8), 110417.

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Immunoblotting Analysis of DNA Damage and Stress Signaling

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Immunoblots were performed as described in [40 (link)]. The following primary antibodies were used: anti γ-H2A.X (phospho-histone H2A.X, sc-101696, Santa Cruz Biotechnology, Heidelberg, Germany), anti-p38MAPK (#9212), anti-phospho-p38 (3D7) (#9215), anti-phospho-p53 (#9284), all obtained from Cell Signaling Technology. For loading control, anti-ribosomal protein L26 (Sigma-Aldrich, Hamburg, Germany) was used. The following secondary antibodies were used: IRDye 680LT goat anti rabbit IgG (926–68021) and IRDye 800CW goat anti-mouse IgG (926–32210) (LI-COR Biosciences, Bad Homburg, Germany). Signals were measured with LI-COR Odyssey^® scanner and quantified with LI-COR Odyssey^® software 3.0 (LI-COR Biosciences).

Schauer M., Kottek T., Schönherr M., Bhattacharya A., Ibrahim S.M., Hirose M., Köhling R., Fuellen G., Schmitz U, & Kunz M. (2015). A mutation in the NADH-dehydrogenase subunit 2 suppresses fibroblast aging. Oncotarget, 6(11), 8552-8566.

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Western Blot Analysis of DDX3X, hnRNPK, and DDX1 Proteins

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Whole protein was harvested with RIPA lysis buffer (Thermo Fisher) freshly supplemented with protease inhibitors (Pierce) and boiled in 1X Laemmli buffer. Samples were run on NuPAGE 10 % Tris-bis gel in 1X MOPS-SDS running buffer. After gel electrophoresis, protein was transferred onto a nitrocellulose membrane using the iBlot transfer kit (Thermo Fisher) according to the manufacturer’s protocol. The membranes were blocked by nutating in 5% BSA in PBST for one hour at room temperature, then stained with primary antibody overnight at 4 °C. Bands were visualized with IRDye secondary antibody staining for one hour prior to imaging on a Licor Odyssey scanner at 700 nm and 800 nm.
Rabbit Anti-DDX3X (Abcam) 1: 5000
Mouse Anti-DDX3X (Abcam) 1: 1000
Mouse Anti-hnRNPK (Abcam) 1: 1000
Rabbit Anti-DDX1 (Proteintech) 1: 1000
Mouse Anti-GAPDH (Abcam) 1: 10,000
IRDye® 680 L T Goat anti-Rabbit IgG (LICOR) 1: 20,000
IRDye® 800CW Goat anti-Mouse IgG (LICOR) 1: 15,000

Cargill M.J., Morales A., Ravishankar S, & Warren E.H. (2021). RNA helicase, DDX3X, is actively recruited to sites of DNA damage in live cells. DNA repair, 103, 103137.

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