β2-GFP knockin dorsal brain regions containing the DRN were dissected and immediately homogenized in the bullet blender tissue homogenizer (NextAdvance, Averill Park, NY, USA), with Co-IP buffer (50 mM Tris, 150 mM NaCl, 0.75% Triton-X 100, Pierce protease inhibitor cocktail). Dynabeads A (ThermoFisher Scientific, Waltham, MA, USA) were pre-incubated with 10μg rabbit anti-GFP primary antibodies (ThermoFisher Scientific,Waltham, MA, USA) and thoroughly washed. Brain homogenates at a concentration of 13 mg·ml-1 were incubated with the dynabeads-antibody complex for 3 days rotating at RT. After washing, GFP fusion protein complexes including interacting proteins were eluted and immediately prepared for western blot analysis.
Dynabeads a
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dynabeads A are uniform, superparamagnetic beads that can be used for the capture and isolation of a variety of biomolecules, including proteins, peptides, nucleic acids, and cells. The beads are coated with Protein A, which binds to the Fc region of antibodies, enabling the selective isolation of antibodies or antibody-bound complexes from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
G dynabeads, by Thermo Fisher Scientific (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
Bullet blender tissue homogenizer, by Next Advance (1 mentions)
Mops buffer, by Thermo Fisher Scientific (1 mentions)
1200 ultrasonic cleaner, by Emerson (1 mentions)
Anti h3k27ac antibody, by Abcam (1 mentions)
Anti igg antibody, by Merck Group (1 mentions)
Faststart universal sybr green master, by Roche (1 mentions)
Anti p27, by Santa Cruz Biotechnology (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Complete protease inhibitor mixture, by Roche (1 mentions)
Ty tag specific antibody, by Thermo Fisher Scientific (1 mentions)
6 protocols using dynabeads a
1
Affinity Purification of GFP-Interacting Proteins
2
FMRP Interactome Isolation from Cerebellum
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Cerebella from WT and Fmr1-KO mice were grinded in liquid nitrogen into fine powder and resuspended in 5 v/w with PBS containing 1% Igepal. Samples were cleared with 15 μl of naked Dynabeads A (Thermofisher) for 30 min at 4°C on a rotating wheel. During this time, 30 μl of Dynabeads A were incubated with anti-FMRP primary antibody for 1 h at room temperature on a rotating wheel, with 100 μg of tRNA, ssDNA and BSA. The “pre-clear” beads were then removed and samples were centrifuged for 10 min at 14,000 rpm at 4°C. Supernatants were incubated with antibody-coated beads overnight at 4°C on a rotating wheel. Beads were washed three times with PBS containing 0.1% Igepal and incubated for 15 min at 55°C with 100 mM dithiothreitol and 2× Laemmli sample buffer. Eluted proteins were then resolved on 4%–12% gradient SDS–PAGE using MOPS buffer (Invitrogen).
3
Chromatin Immunoprecipitation (ChIP) Protocol
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For chromatin immunoprecipitation (ChIP), cells were harvested and fixed after stimulation. Samples were lysed and sonicated using a Branson 1200 Ultrasonic Cleaner to obtain chromatin fragments of optimal size (300-800 bp). Sheared chromatin was incubated using 2 μL of anti-H3K27ac antibody (Abcam, ab4729) or 2 μL of anti-IgG antibody (Millipore, 12-370), and immunoprecipitated with Dynabeads A (Invitrogen, 10001D) and Dynabeads G (Invitrogen, 10003D) magnetic beads. ChIP samples were de-cross-linked with proteinase K (Thermo Fisher Scientific) at 65°C for 4 h. Then, DNA was purified with the DNA Clean Kit & Concentrator TM-5 (Zymo Research). Quantitative analysis of the purified ChIP and Input DNAs was performed by using FastStart Universal SYBR Green Master (Roche) by real time qPCR.
4
ChIP Assays with Gingival Fibroblasts
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ChIP assays were performed by crosslinking ∼5 million cells (Gingival fibroblasts; AG09319, Corriell Institute). Four μg of antibody for H3K4me1 (Abcam ab8895 lot GR61294-1) and H3K27ac (Abcam ab4729 lot GR183919-2) were coupled to 10 μL of Dynabeads A and 10 μL of Dynabeads G (Invitrogen 10001D and 10004D, respectively) per ChIP (See Supplemental Methods ).
5
Immunoprecipitation and Immunoblotting of Cell Signaling Proteins
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Proliferating MEFs and C2C12 cells were scraped and washed twice with PBS. Pellets were lysed in 1 ml of IP buffer (PBS containing 0.5% Triton X-100, 1 mM EDTA, 100 μM sodium orthovanadate, 0.25 mM PMSF, complete protease inhibitor mixture by Roche Applied Science, and 1/25 vol of DNAse I (Sigma). Cell lysates were incubated on ice for 30 min and then centrifuged at 3000 rpm at 4°C for 5 min. After centrifugation, samples were quantified using Lowry method [36 (link)]. 1 mg of total protein was incubated overnight at 4°C with 2 μg of anti-p27 (Santa Cruz SC528). Samples were incubated with A-Dynabeads (Invitrogen) for 2 h and the immunocomplexes were extensively washed with IP buffer, subsequently eluted with 0.1 M Citrate pH 2.5 and boiled at 100°C in Laemmli buffer for Immunobloting analysis. As a control, lysates were incubated with irrelevant rabbit IgG. Antibodies used for Immunoblotting were: anti-p27 (BD Transduction Laboratories 610242; 1:1000), anti-Pax5 (Santa Cruz SC1974; 1:500), anti-MyoD (Santa Cruz SC760; 1:500) and anti-AHR (ENZO life Sciences BML-SA210-0100; 1:2000).
6
Ty Tag-Specific Antibody Immune Capture
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Immune capture experiments were performed using a Ty tag-specific antibody (ThermoFisher Scientific). A whole-cell extract was created with Ty-TbMex67 and RNAi TbMtr2-TyTbMtr2 (uninduced) as previously described (33 (link)). Antibody cross-linked protein A-Dynabeads (Invitrogen) were pretreated with poly(dI-dC) for 1 h at 4°C and then were incubated with extract (1 mg/ml) overnight at 4°C. The beads were washed three times with phosphate-buffered saline (PBS) containing 0.05% Tween 20. Beads were then heated at 70°C for 10 min with SDS-PAGE sample buffer to elute off bound proteins. Western blot analysis was performed as described below, and results shown are representative of three biological replicates.
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