Filter retardation assay (FRA) was performed as previously described (Ast et al., 2018b (link)), utilizing a final protein concentration of 1.5 μM. The addition of PP started the aggregation process. At the indicated time points at 0, 4, 8 and 24 h after addition of PP, an aliquot was taken from each sample and flash frozen in liquid nitrogen. Before blotting, samples were mixed 1:1 v/v with a stop solution (4% SDS, 100 mM DTT). In a dot blot apparatus attached to a pump, whole samples were loaded on and filtered simultaneously through a cellulose acetate membrane with 0.2 μm pores (OE66, GE Healthcare, #10404180) pre-soaked in TBS buffer containing 1% SDS. All blot wells were washed twice with TBS plus 0.1% SDS and membranes were then blocked in 5% skim milk in TBS-T. Fibrils retained on the membrane were detected by probing with both anti-HTTEx1 primary antibodies EPR5526 (1:1000; Abcam) and MW8 (1:2000; DSHB). Anti-rabbit HRP secondary antibody (1:10,000; #31460, Thermo Fisher) was used to probe HTTEx1 EPR5526 and IRDye680 anti-mouse (1:10.000; #926-68070 LI-COR Biosciences) was used for detection of MW8. HRP signals were developed with Pierce ECL Western Blotting Substrate (#32209, Thermo Fisher) and imaged using the OdysseyFC imaging system (LI-COR Biosciences).
Irdye680 anti mouse
Manufactured by LI COR
Sourced in United States
The IRDye680 anti-mouse is a fluorescent-labeled antibody designed for use in immunoassays and other applications that require the detection of mouse antigens. The dye is IRDye680, which absorbs and emits light in the near-infrared region of the spectrum, making it suitable for use in a variety of detection platforms.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit irdye 800, by LI COR (6 mentions)
Odyssey imaging system, by LI COR (3 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Phosstop, by Roche (2 mentions)
Epr5526, by Abcam (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Rabbit anti c myb h141, by Santa Cruz Biotechnology (1 mentions)
Mouse anti gapdh, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Proteintech (1 mentions)
P0013, by Beyotime (1 mentions)
Total protein extraction buffer, by Beyotime (1 mentions)
Krt17, by Proteintech (1 mentions)
Cyclin d1, by Merck Group (1 mentions)
Cyclin e, by Merck Group (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Proteintech (1 mentions)
Itga3, by Abcam (1 mentions)
P src tyr527, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ir dye 800 anti rabbit immunoglobulin g igg secondary antibodies, by LI COR (1 mentions)
11 protocols using irdye680 anti mouse
1
Fibril Aggregation Detection via Filter Retardation
2
Immunoblot Detection of c-Myb and GAPDH
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For immunoblot detection, the following antibodies were used: rabbit anti‐c‐Myb H141 (sc7874; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti‐GAPDH (AM4300; Invitrogen, Thermo Fisher Scientific), IRDye800 anti‐rabbit (926‐32213; LI‐COR Biosciences, Lincoln, NE, USA) and IRDye680 anti‐mouse (926‐68072; LI‐COR Biosciences).
3
Western Blot Analysis of CCBE1 Protein
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Fresh GIST tissues were lysed in tissue protein extraction reagent (Invitrogen). Primary GIST cells were lysed in Western and IP lysis buffer (P0013, Beyotime, Jiangsu, China) supplemented with 1 mM PMSF (Adamas beta, Shanghai, China). Equal amounts of protein were loaded and separated through SDS-PAGE. The proteins were transferred to a membrane, and the blot was blocked with 10% non-fat milk in TBS. The membranes were incubated overnight at 4 °C using the following antibodies: CCBE1 (Sigma Life Science, USA, HPA041374, 1:1000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech Group, Chicago IL). After incubating with the IRDye 680 anti-mouse (LI-COR, Lincoln, NE) and IRDye 800 anti-rabbit (LI-COR, Lincoln, NE) secondary antibodies for 1 hour at room temperature, the bands were detected by an Odyssey infrared imaging system (LI-COR, Lincoln, NE).
4
Western Blot Analysis of Apoptosis Regulators
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Total protein of cells and tissue were extracted using a total protein extraction buffer (Beyotime, China). Cell lysates were separated by 10% SDS-PAGE gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were blocked with 5% nonfat milk and incubated with the primary antibodies and then incubated with species-specific secondary antibodies. Image J software was used to quantify the results. Antibodies and their corresponding concentrations used were as follows: Krt17 (1:1000, Proteintech, USA), Bcl2 (1:1000, CST, USA), Bax (1:1000, CST, USA), Bcl-xl (1:1000, CST, USA), Cleaved caspase-3 (1:1000, CST, USA), Cyclin D1 (1:1000, Sigma, USA), Cyclin E (1:1000, Sigma, USA), IRDye680 anti-mouse (1:20000, LI-COR) and IRDye800 anti-rabbit (1:10000, LI-COR). β-actin (1:1000, CST, USA) was used as a control to ensure the equal loading of protein.
5
Whole Cell Lysate Preparation and Protein Detection
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Whole cell lysates were prepared by lysis buffer (50mM Tris-HCl, 150mM NaCl, 1% Triton-X 100, 1mM MgCl2, MnCl2 and CaCl2, 1mM PMSF and 10mM sodium fluoride) [49 (link)]. The primary antibodies used included the following: DPT (HuaAn Biotechnology, Hangzhou, China), focal adhesion kinase (FAK) (Abcam, Cambridge, UK), p-FAK Tyr397 (Abcam, Cambridge, UK), Src (Cell Signaling Technology, Boston, MA), p-Src Tyr527 (Cell Signaling Technology, Boston, MA), ITGA3 (Abcam, Cambridge, UK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech Group, Chicago IL). After incubating with the IRDye 680 anti-mouse (LI-COR, Lincoln, NE) and IRDye 800 anti-rabbit (LI-COR, Lincoln, NE) secondary antibodies for 1 hour at room temperature, the bands were detected by an Odyssey infrared imaging system (LI-COR, Lincoln, NE). Quantification was analyzed by using Image J software.
6
Western Blot Analysis of AFMID and TP53
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Cells were washed with PBS and lysed in RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 1% [v/v] NP40, 0.5% [w/v] deoxycholate, 0.1% [w/v] SDS) supplemented with Complete Protease Inhibitor Cocktail tablets (Roche) and PhosSTOP (Roche). Thirty micrograms of total protein were loaded on a 12% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Millipore). For AFMID detection, the membrane was blocked in 1% (w/v) BSA in Tween 20-TBST (50 mM Tris pH 7.5, 150 mM NaCl, 0.05% [v/v] Tween 20). For TP53 detection, the membrane was blocked in 5% (w/v) milk in Tween 20-TBST. Blots were incubated with AFMID (Proteintech, 19522-1-AP), Tubulin (Sigma, T9026), or TP53 (Santa Cruz, sc-126) primary antibodies. IR-Dye 680 anti-mouse or IR-Dye 800 anti-rabbit immunoglobulin G (IgG) secondary antibodies (Licor) were used for infrared detection and quantification with an Odyssey Imaging System (Licor).
7
Western Blot Analysis of Protein Targets
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Cells lysates were prepared by RIPA buffer containing protease inhibitors. Denatured lysates were separated by 10% SDS-PAGE gel electrophoresis and transferred onto PVDF membranes (Millipore Corp, Billerica, MA, USA). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies overnight at 4°C. The following antibodies were used: anti-TUFT1 (1: 500 dilution, Santa Cruz), anti-phospho-CREB1 (Ser133, 1 : 1000, ImmunoWay), anti-ZYX (1 : 1000, Proteintech), and anti-β-actin (1 : 1000 dilution, Santa Cruz). After washing with TBS/Tween-20, the membranes were incubated with IRDye 680 anti-mouse or IRDye 800 anti-rabbit (LI-COR) secondary antibodies for 1 hr at room temperature. The bands were detected by Odyssey imaging system (LI-COR).
8
Prostate Cancer Antigen 3 Virus Assay
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22Rv1 cells (1.6 × 105/well) were seeded 24h prior to infection with PCA3-fl, PCA3-TSTA and PCA3-3STA. Relative luciferase unit (RLU) was assayed at the indicated time points for each virus. Western blot analysis was performed on the collected lysates with 4μg of protein and the membranes were incubated with antibodies against β-actin (1:5000, Sc47778, Santa Cruz, CA, USA) or vp16 (1:5000, ab4809, Abcam, Toronto, ON, Canada). The secondary antibodies (1:5000) were conjugated with IRDye™ 680-anti-mouse and IRDye™ 800-anti-rabbit antibodies (LI-COR, Lincoln, NE, USA). Proteins were detected and the signal was quantified by means of the Odyssey Scanning System (LI-COR, Lincoln, NE, USA).
9
Protein Expression Analysis via Western Blot
10
Western Blot Analysis of Apoptosis-Related Proteins
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Total cellular proteins were extracted using a total protein extraction kit (Beyotime, China). Cell lysates were separated by 10% SDS-PAGE gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were blocked with 5% nonfat milk and incubated with the primary antibodies and then incubated with species-specific secondary antibodies. Image J software was used to analyze the results. The following antibodies were used at the indicated concentrations PEA15 (1:1000, Proteintech, USA), Bcl2 (1:1000, CST, USA), Bax (1:1000, CST, USA), Cleaved caspase-3 (1:1000, CST, USA), ERK(1:1000, Abcam, USA), p-ERK(1:1000, Abcam, USA), IRDye680 anti-mouse (1:20000, LI-COR) and IRDye800 anti-rabbit (1:10000, LI-COR). GAPDH (1:1000, CST, USA) was used as a control to confirm equal loading of protein samples.
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