Mouse cerebellar tissues and cell pellets were homogenized with a cell lysis buffer (Cell Signaling) with Halt phosphatase inhibitor (Thermo-Fisher), complete protease inhibitors (Roche) and PSMF (Sigma-Aldrich). The supernatant was recovered by centrifugation (16,000g, 15mins). Protein concentration was assayed by the Bradford protein assay system (Bio-rad). Twenty micrograms of lysates were loaded into 4–12% Bis—Tris gels (Invitro-gen). Electrophoresis was carried out according to the manufacturer’s recommendations. Following electrophoresis, the proteins were transferred to nitrocellulose membranes by the iBlot device (Invitrogen), blocked with an Odyssey blocking buffer (LI-COR Biotechnology) for 1 h. Membranes were incubated overnight with the following primary antibodies in blocking buffer: Iba1(Wako), CD11b(Abcam), MUTYH(Abcam), PARP-1(Trevigen), Frataxin (Santa Cruz), Actin(Abcam), AT1(Abcam), Tubulin (Abcam). Subsequently, the membranes were incubated with a corresponding pair of IRDye 680CW and IRDye 800CW-coupled secondary antibodies (LI-COR). Proteins were visualized with the Odyssey infrared imager and software (LI-COR) according the manufacturer’s instruction.
Mutyh
Manufactured by Abcam
MUTYH is a DNA glycosylase enzyme that is involved in the base excision repair pathway. It is responsible for the removal of adenine and 2-hydroxyadenine from DNA, helping to maintain genomic integrity.
Automatically generated - may contain errors
Lab products found in correlation
Tubulin, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Irdye 680cw, by LI COR (1 mentions)
Parp1, by Bio-Techne (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Halt phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Iblot device, by Thermo Fisher Scientific (1 mentions)
Frataxin, by Santa Cruz Biotechnology (1 mentions)
Irdye 800cw coupled secondary antibodies, by LI COR (1 mentions)
Odyssey infrared imager and software, by LI COR (1 mentions)
Bradford protein assay system, by Bio-Rad (1 mentions)
Cd11b, by Abcam (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Tubulin, by Abcam (1 mentions)
Actin, by Abcam (1 mentions)
Iba1 antibody, by Fujifilm (1 mentions)
Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (1 mentions)
Cd11b, by Bio-Rad (1 mentions)
5 protocols using mutyh
1
Western Blot Analysis of Cerebellar Proteins
2
Immunostaining of Mouse Brain Cerebellar Tissue
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Mice were perfused with 4% paraformaldehyde in 0.1M PBS at one day or 7 day time point after stereotaxic surgeries. 10μm brain sections from cerebellum region were processed with cryostat. Fluorescence immunostainings were performed using Alexa Fluor 488- and Alexa Fluor 647- tagged secondary antibodies (Invitrogen). The following primary antibodies were used: Iba-1 antibody (1:100, Wako), CD11b (1:100, Abdserotec), MUTYH (1:500, Abcam), PARP-1 antibody (1:1000, Trevigen) and GFAP (1:500, Cell signaling). Primary antibodies were incubated overnight at 4°C followed by secondary antibody incubation two hours at room temperature. Fluorescent microscopy was performed with Retiga 2000R camera (Qimaging) mounted on a Nikon microscope (Nikon, USA). For statistical analyses, fluorescent immunostaining intensity was measured with ImageJ software (NIH) and cells were counted from 6 coronal sections per mouse.
3
Comprehensive Western Blotting for Protein Analysis
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Western blotting was performed as previously described46 (link). Electron transport proteins were detected using a total OXPHOS human western blot antibody cocktail (ab110411) from Abcam. The primary antibodies used are SFXN4 (Thermofischer (PA5-35980)), P-Histone H2A.X (Cell Signaling Technology (9718S)), Histone H2A.X (Cell Signaling Technology (2595S)), XPD (D3Z6I) (Cell Signaling Technology (11963)), NTH1 (Abcam (ab236912)), MUTYH (Abcam (ab228722)), FANCJ (BACH1/BRIP1) (Cell Signaling Technology (4578)), POLD1 (BD Biosciences (610972)), FTH46 (link), TFR1 (Thermo Fisher (13–6800)), HSP60 (Cell Signaling Technology (12165S)), AFG3L2 (Proteintech (14631-1-AP)), HSP90 (Cell Signaling Technology (4877), Tubulin (Cell Signaling Technology (12351)), β-Actin (Sigma Aldrich (A3854) C/EBPβ Antibody (Cell Signaling Technology #9008),Id2 (D39E8) (Cell Signaling Technology #3431) and RPA32/RPA2 Antibody (Cell Signaling Technology #52448).
4
Immunoblotting Using RIPA Lysis and ECL Detection
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For immunoblotting, the cells were lysed in RIPA buffer containing protease inhibitors (Bimake; No. B14002) and phosphatase inhibitors (Bimake; No. B15003). Protein concentrations were detected by bicinchoninic acid assay (Yeasen Biotechnology; No. 20201ES90). Then, the proteins were resolved by SDS-PAGE gel and transferred to PVDF membranes (EMD Millipore; No. IPVH00010). After blocking with 5% bovine serum albumin (Sigma; No. V900933-1KG), the membranes were incubated at 4 °C overnight with the indicated primary antibodies, including Flag (1:5000, Sigma; No. GNI4110-FG), MUTYH (1:1000, Abcam; No. ab228722) and Vinculin (1:3000, Sigma; No. V9131). After washing with PBS three times the next day, the membranes were incubated with horseradish peroxidase-conjugated mouse or rabbit secondary antibodies (1:5000, Cell Signaling Technology; No. 7076V and 7074V, respectively). Finally, the protein bands were visualized using an enhanced chemiluminescence substrate kit (Yeasen Biotechnology; No. 36208ES80).
5
Protein Extraction and Western Blot Analysis
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Cells were lysed in lysis buffer composed of RIPA lysis buffer supplemented with protease inhibitors (Sigma) and phosphatase inhibitors (Sigma, NEB). After sonication and centrifugation of the lysates, they were heated with reducing sample buffer. Protein samples were separated by SDS-PAGE (3%-8% or 4%-12% gradient gels; Invitrogen) and then transferred onto nitro-cellulose membranes. All primary antibodies were used at 1:1,000 dilution and secondary antibodies at 1:5,000. Antibodies used were: XPA (14607S; Cell Signaling), MUTYH (ab55551; Abcam), PARP (9532; Cell Signaling), P-S140-H2AX (07-164; Millipore), pS4/ S8-RPA (A300-245A-T; Bethyl Laboratories), TUBULIN (3873, Cell Signaling) and b-ACTIN (A 5060A; Sigma).
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