1 mg of cell lysate generated from MDA‐MB‐231 cells as described above was diluted to 500 µL in TBST in the presence or absence of UNC6261 or UNC7394 and allowed to spin at 4 °C overnight. Magnetic streptavidin M‐280 Dynabeads (30 µL beads per pulldown) were incubated with UNC6261‐Biotin (1 µL of 10 mm stock) in TBST. The beads were left to rotate for 45 min at room temperature. Next, the Dynabeads were washed with TBST to remove excess biotin ligand and then added to the lysate solution. The mixture was rotated at 4 °C overnight, and then the beads were isolated by magnetization and washed with TBST. Beads were then resuspended with 15 µL TBST and 15 µL 2× LaemmLi sample buffer and heated at 95 °C for 5 min. In parallel, input of samples 10–15 µg of cell lysate was diluted to 7.5 µL and 2× Laemmi sample buffer was added and heated at 95 °C for 5 min. Half of each pull down sample was used for analysis by SDS‐PAGE (BioRad any kD) and Western blotting. Following membrane transfer, membranes were incubated with primary antibody against CDYL (1:1000, Abcam ab5188) at 4 °C overnight, washed with TBST, and then treated with the complimentary secondary antibody‐IRDye conjugate (Li‐COR; 1:10000 TBST) for 1 h at room temperature and visualized on a Li‐COR Odyssey instrument.
Odyssey instrument
Manufactured by LI COR
Sourced in United States
The Odyssey instrument is a lab equipment product designed for fluorescence detection and quantification. It features high-sensitivity detection capabilities and supports a range of fluorescent labeling dyes. The Odyssey instrument is suitable for various applications, including Western blotting, protein and cell-based assays, and more.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Irdye conjugated secondary antibody, by LI COR (4 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Odyssey blocking buffer, by LI COR (3 mentions)
Bsm 33033m, by Bioss Antibodies (3 mentions)
Protease inhibitor and phosphatase inhibitor cocktail, by Roche (3 mentions)
Immobilon p transfer membrane, by Merck Group (3 mentions)
Anykd, by Bio-Rad (2 mentions)
Actin, by Cell Signaling Technology (2 mentions)
Protein kit, by Bio-Rad (2 mentions)
Anti ccl20, by Biorbyt (2 mentions)
Dual color, by Bio-Rad (2 mentions)
Ab8226, by Abcam (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Tnt t7 quick coupled transcription translation system, by Promega (2 mentions)
Gfp 3e6 a11120, by Thermo Fisher Scientific (2 mentions)
50 protocols using odyssey instrument
1
Affinity Purification of CDYL from MDA-MB-231 Cells
2
Exosome Lysis and Protein Analysis
3
Western Blot Analysis of ADAM17 and PEDV N
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Western blot analysis was performed as previously described with a slight modification (Gu et al., 2015 (link)). Typically, samples were separated by SDS-PAGE under reducing conditions and transferred onto a PVDF membrane. After blocking, the membranes were incubated with a primary antibody and then incubated with an appropriate IRDye-conjugated secondary antibody (Li-Cor Biosciences, Lincoln, NE). The membranes were scanned using an Odyssey instrument (Li-Cor Biosciences) according to the manufacturer's instructions. The anti-ADAM17 polyclonal antibody was purchased from Abcam (Abcam, Cambridge, MA). Mouse anti-Flag antibody was purchased from Sigma (Sigma-Aldrich, USA) and used to detect overexpression of ADAM17 fused with a Flag tag in the carboxyl terminal. Mouse mAb 2G3 against PEDV N protein was stocked in our laboratory. Anti-β-actin mAb was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
4
Western Blot Analysis of SMN Protein
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Twenty milligrams of each mouse tissue was pulverized in liquid N2 and homogenized in 0.4 ml of protein sample buffer containing 2% (w/v) sodium dodecyl sulphate (SDS), 10% (v/v) glycerol, 50 mM Tris–HCl (pH 6.8) and 0.1 M DTT. Protein samples were separated by 12% SDS-polyacrylamide gel electrophoresis and electro-blotted onto nitrocellulose membranes. The membranes were blocked for 2 h with 5% (w/v) non-fat milk in Tris-buffered saline containing 0.05% Tween-20 (TBST), and then incubated overnight at 4°C with primary antibodies: monoclonal anti-SMN (BD Biosciences, 1:500) and anti-α-tubulin (Sigma). After washing with TBST for three times, the membranes were incubated with secondary IRDye 700CW-conjugated goat anti-mouse or anti-rabbit antibody. Protein signals were detected with an Odyssey instrument (LI-COR Biosciences).
5
Cell Lysis and Protein Analysis
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For protein analysis, cleared NP40 lysates and whole cell SDS lysates were prepared from 10-cm dishes containing cells at 100% confluency as described [17 (link), 26 (link)]. Protein concentration was measured by BCA assay and 50 micrograms protein was loaded per lane. Immunoprecipitations were performed as described except that the buffer contained 0.5% NP40 [11 (link)]. Antibodies for E-cadherin, N-cadherin, beta catenin, phospho-beta catenin, beta four integrin, and fibronectin were all rabbit polyclonal from Cell Signaling; for Myc-tag (clone 9E10.3), from Neomarkers; for FLAG tag (clone M2), Agilent; for actin (125-ACT), from PhosphoSolutions; for GFP, from Abcam; for vimentin, from Millipore. ZO-1 antibody was from PTG. The CLCA2 antibody TVE20 has been described (12). The protein size marker was Dual color (Bio-Rad). Secondary antibodies were labeled with IR680 or IR800 (Licor), and protein expression was quantified on an Odyssey instrument (Licor).
6
Western Blot Analysis of Exosomal Proteins
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Western blots were performed by separating 20 μg of protein in 1× RIPA buffer by 10% SDS-PAGE. Samples treated with 2.5 mU peptide-N-glycosidase F (Sigma Aldrich) were incubated for 3 h at 37 °C prior to SDS-PAGE separation. Protein bands were transferred to polyvinylidene fluoride (PVDF) membranes by wet transfer at 100 V for 1 h. The membranes were blocked with 5% non-fat dry milk in TBST and 10% goat serum and incubated with primary antibodies (Hsp70, CD81, CD63, CD9, System Biosciences) at 4 °C overnight. The membranes were washed in a mixture of tris-buffered saline and polysorbate 20 (TBST) and incubated in goat-anti rabbit IgG-horseradish peroxidase (HRP) secondary antibody (Systems Biosciences) at room temperature for 1 h. The protein bands were visualized using the ECL Plus Substrate solution (Pierce) and imaged using an Odyssey instrument (Licor Biotechnology).
7
Screening for Huntingtin Interactors
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The unbiased screen for htt protein interactors was performed using a Panomics transcription factor (TF) immunoprecipitation array, according to the manufacturer’s instructions: http://www.veritastk.co.jp/attached/2209/TFInteractionArrayKitcombined.pdf . Htt IP for the Panomics TF IP array utilized anti-htt antibody EM48. Protein lysates were prepared as previously described55 (link). For co-immunoprecipitations, homogenized protein was incubated with indicated co-immunoprecipitation antibody (GFP: 3E6 A11120, Life Technologies (1:100); Htt: MCA2050, Abcam (1:100); PPARδ: SC-7197, Santa Cruz; (1:100)). Proteins were run on 10% Bis-Tris gels (Invitrogen) and transferred to PVDF membranes (Millipore) prior to blocking in Odyssey Blocking Buffer (LI-COR biosciences). Membranes were incubated with antibodies as indicated: Flag (F7425, Sigma; 1:1000), Htt (MAB2166, Millipore; 1:1000), GFP (A11121, Life Technologies, 1:1000), PPARδ (PA1-823A, Pierce; 1:1000), PPARα (PA1-822A, Pierce; 1:1000), or PPARγ (PA3-821A, Pierce; 1:1000), or β-actin (ab8226, Abcam; 1:10,000), and imaged on the Odyssey instrument (Licor). To obtain proteins in vitro for the direct interaction co-immunoprecipitation, the Promega TnT T7 Quick Coupled Transcription/ Translation System (Promega, #L1170) was used prior to co-immunoprecipitation.
8
Northern Blot RNA Detection Protocol
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For northern blot, total RNA was harvested by using Trizol reagent (Invitrogen, USA) and analyzed by agarose-formaldehyde gel electrophoresis. RNAs were transferred to a 0.45-μm nylon membrane and probed with biotin-labeled DNA probes generated with the specific primers (Table 1 ) using the North2SouthTM biotin random prime DNA labeling kit (Thermo Scientific). The membrane was imaged an Odyssey instrument (Li-Cor Biosciences) followed by incubation with IRDye 800-conjugated streptavidin.
9
Western Blot Analysis of Muscle Proteins
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Ice-cold radio immunoprecipitation (RIPA) lysis buffer (Beyotime, Shanghai, China) with 1 mM phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China) was used in the total protein extracted from gastrocnemius muscle tissues or CPM cells. SDS-page gel (10%) was used to separate proteins, and the separated proteins were transferred to the polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). After 30 min of blocking, the membrane was incubated with anti-MYHC (1:200; B103, DSHB) or anti-GAPDH (1:2000; bsm-33033M, BIOSS) at 4 °C for 12 h. Anti-mouse secondary antibody (1:10,000; 7076P2, CST) was used to incubate the membranes. The ECL Peroxidase Color Development Kit (Beyotime, Shanghai, China) was used in chromogenic reactions by following the manufacturer’s protocol. Protein band visualization was performed using the Odyssey instrument (Li-cor, Lincoln, NE, USA).
10
Western Blot Analysis of Muscle Protein Expression
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Total protein was released from cells through lytic effect by using ice-cold radioimmunoprecipitation (RIPA ) lysis buffer (Beyotime, Shanghai, China) with 0.5 mM/mL phenylmethylsulfonyl fluoride (Solarbio, Beijing, China). Lysate was centrifuged at 13,000 × g at 4°C for 15 min. The protein supernatant was transferred in a new tube and was incubated in 98°C with Protein Buffer (TransGen Biotech, Beijing, China) for 5 min. Same amount of protein was separated in a 12% SDS-PAGE, and transferred to PVDF membranes (BioRad, CA). QuickBlock Blocking Buffer (Byotime, Shanghai, China) was used to membranes incubation for 30 min at room temperature. Blocked membranes were incubated with antibodies against MyoD (1:500, NovusBio, CO), MyHC (1:200, DHSB, IA), MurF-1 (1:500, Proteintech, IL), Atrogin-1 (1:1000, Abcam, Cambridge, UK) and GAPDH (1:2000, Bioworld, MN) overnight at 4°C. Next, anti-mouse or anti-rabbit IgG secondary antibodies with HRP label were used to incubate the membranes for 1 h at room temperature. High sensitivity ECL chemiluminescence detection kit (Vazyme, Nanjing, China) was used in chromogenic reaction by following the instruction. Chromogenic bands were captured in an Odyssey instrument (Li-cor, CA). The gray value of bands was measured in ImageJ software.
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