Gp62 c

Manufactured by Merck Group

The GP62-C is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use, providing a core function for specific applications. The details of its intended use or performance characteristics are not available in this factual, unbiased description.

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Lab products found in correlation

Mouse anti β actin, by Merck Group (2 mentions) Protease inhibitor, by Roche (1 mentions) Nitrocellulose membrane, by Cytiva (1 mentions) Nb100 2220, by Progen Biotechnik (1 mentions) Np0322box, by Thermo Fisher Scientific (1 mentions) Sc 32282, by Santa Cruz Biotechnology (1 mentions) Parkin, by Santa Cruz Biotechnology (1 mentions) Mthsp70, by Thermo Fisher Scientific (1 mentions) Ab3243, by Abcam (1 mentions) Htra2, by R&D Systems (1 mentions) Neuronal nuclei (neun), by Merck Group (1 mentions) Fluoromount, by Merck Group (1 mentions) Dfc9000 gt camera, by Leica (1 mentions) Mab377, by Proteintech (1 mentions) Tdp 43, by Proteintech (1 mentions) Gtx100042, by Abcam (1 mentions)

4 protocols using gp62 c

Quantitative Protein Analysis of Differentiated TR5TY6 Cells

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Differentiated TR5TY6 cells induced for 0, 1, 3, and 6 days were homogenized in RIPA buffer supplemented with protease inhibitors (Roche) and cell extracts clarified by centrifugation at 10,000 × g for 10 min at 4 °C. Protein concentrations were quantified using the BCA method and subjected to SDS-PAGE. Proteins were resolved in 10 or 15% polyacrylamide gels and transferred onto 0.45 µm nitrocellulose membranes (Amersham). Blocking with 5% milk powder in PBS was followed by overnight incubation at 4 °C with the primary antibodies mouse anti-tyrosinase (1:2000, Thermo Fisher Scientific, Cat#MS-800-P1), rabbit anti-Lamp1 (1:250, GeneTex, Cat#GTX19294), rabbit anti-LC3 (1:1000, Novus Biologicals, Cat#NB100-2220), guinea pig anti-p62 (1:1000, Progen, Cat#GP62-C) or mouse anti-β-actin (1:5000, Sigma-Aldrich, Cat#A5441). Incubation with the secondary antibodies donkey anti-rabbit, sheep anti-mouse (both 1:5000, from Amersham), and goat anti-guinea pig (1:1000, Santa Cruz Biotechnology) was performed for 1 h at RT. Band densitometry, normalized to β-actin expression, were measured using ImageJ image analysis software.

Carballo-Carbajal I., Laguna A., Romero-Giménez J., Cuadros T., Bové J., Martinez-Vicente M., Parent A., Gonzalez-Sepulveda M., Peñuelas N., Torra A., Rodríguez-Galván B., Ballabio A., Hasegawa T., Bortolozzi A., Gelpi E, & Vila M. (2019). Brain tyrosinase overexpression implicates age-dependent neuromelanin production in Parkinson’s disease pathogenesis. Nature Communications, 10, 973.

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Western Blot Protein Analysis

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Cell lysates denatured in SDS were separated on 4–12% Bis-Tris gels (Novex, NP0322BOX). Proteins were transferred on to PVDF membranes and probed using guinea pig anti-SQSTM1/p62 (PROGEN Biotechnik, GP62-C) and Mouse anti-β-Actin (Sigma, AC-15). Mouse 800 (LI-COR 926-32210) and guinea pig 680 (LI-COR 926-3241) were visualized with a LI-COR Odyssey Infrared imager (LI-COR Cambridge, UK).

Cottam E.M., Whelband M.C, & Wileman T. (2014). Coronavirus NSP6 restricts autophagosome expansion. Autophagy, 10(8), 1426-1441.

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Immunofluorescence Analysis of Mitochondrial Proteins

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Antibodies against PGC1α (Millipore Ab3243), TFAM (Abcam Ab119684), NEF2L2 (Abcam ab31163), ClpP (Sigma HPA010649), Htra2 (R&D Systems AF1458), mtHsp70 (Thermofisher), Hsp60 (BD Transduction Systems 611562), GPS2 21 (link), p62 (Progen GP62-C), Beclin1 (Millipore Ab15417), Pink1 (Abcam Ab23707), LC3-II (cell signaling 4108) and Parkin (Santa Cruz biotechnologies sc-32282) to assess signaling, were used in combination with MTCOI, SDHA and DAPI to assess changes with COX-deficiency in a single section of muscle. Antibodies that did not produce a sufficient signal-to-noise ratio, or were highly correlated with mitochondrial mass, were removed from further analysis. TFAM, Hsp60, GPS2 and Beclin1 were selected for immunofluorescence on serial sections (n = 4) of patients with multiple mtDNA deletions (n = 3, patients 4, 5 and 7, Supplementary Table 1). IMARIS v.7.7.2 (bitplane) was used to assess average fluorescent intensity of TFAM and Hsp60 relative to MTCOI/SDHA ratio in whole COX-positive and COX-deficient fibers. Subsequently average intensity of the foci was compared to a COX-positive region of the fiber.

Vincent A.E., Rosa H.S., Pabis K., Lawless C., Chen C., Grünewald A., Rygiel K.A., Rocha M.C., Reeve A.K., Falkous G., Perissi V., White K., Davey T., Petrof B.J., Sayer A.A., Cooper C., Deehan D., Taylor R.W., Turnbull D.M, & Picard M. (2018). Subcellular origin of mitochondrial DNA deletions in human skeletal muscle. Annals of Neurology, 84(2), 289-301.

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Immunofluorescence Staining of Brain Sections

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Following deep/lethal anesthesia, animals were PBS perfused. One brain hemisphere was fixed overnight in 4% PFA in PBS (pH = 7.4) and transferred to 30% sucrose in PBS. Brains were processed into 35‐μm‐thick sagittal sections. Sections were blocked in 4% goat serum and incubated with indicated antibodies of the bellow listed primary antibodies. After several washing steps, sections were incubated with fluorophore conjugated secondary antibodies as indicated (Alexa 488, 594, 647; Thermo Fisher Scientific). After additional washing steps, nuclei were visualized with DAPI. Sections were embedded in Fluoromount™ (Sigma‐Aldrich, Merck). Image acquisition was performed on a LEICA DMI‐8 with a DFC9000GT camera and the following objectives: (HC PL FL L 20×/0.40 CORR PH1; HC PL APO 40×/0.95 CORR). Confocal Images were acquired on Zeiss LSM 800 Microscope Axio Observer 7 (Objective Plan‐Apochromat 63×/1,4 Oil M27). The following antibodies were used for immunofluorescence: GFAP (DAKO, Z0334), IBA1 (GeneTex, GTX100042), CD68 (Abcam, ab125212), CLEC7A (R&D Systems, AF1756), TREM2 (R&D Systems, AF1729), p62 (MBL, PM045), p62 (Progen Biotech GP62C), NeuN (Millipore, MAB377), TDP‐43 (Proteintech, 12892‐2AP), and Phospho‐TDP‐43 409/410 (Cosmo Bio Co, CAC‐TIP‐PTD‐M01).

Werner G., Damme M., Schludi M., Gnörich J., Wind K., Fellerer K., Wefers B., Wurst W., Edbauer D., Brendel M., Haass C, & Capell A. (2020). Loss of TMEM106B potentiates lysosomal and FTLD‐like pathology in progranulin‐deficient mice. EMBO Reports, 21(10), e50241.

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