Samples (50 μg) were mixed with bromophenol blue and 2-mercaptoethanol, boiled for 10 min, electrophoresed on 10% SDS polyacrylamide gel and electroblotted onto a PVDF transfer membrane (Millipore, Billerica, MA, USA). The membrane was blocked with PBS containing 5% skimmed milk, 0.05% Tween 20 and then incubated for 1 h with rabbit polyclonal antibodies to PPAR-γ (sc-7196; Santa Cruz Biotechnologies, Inc., Dallas, TX, USA), and mouse monoclonal antibodies to GAPDH (MAB374; Millipore), which were diluted to 1 : 500 with blocking buffer. After washing, the membrane was incubated with 1 : 5000 dilution of horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG or HRP-conjugated donkey anti-mouse IgG (GE Healthcare, Little Chalfont, UK) in blocking buffer. Subsequently, the blots were developed using the ECL detection kit (GE Healthcare) and protein bands were visualized using the VersaDoc system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Hrp conjugated donkey anti mouse igg
Manufactured by GE Healthcare
Sourced in United Kingdom, Japan
HRP-conjugated donkey anti-mouse IgG is a secondary antibody conjugated with horseradish peroxidase (HRP). It is used to detect and quantify mouse immunoglobulin G (IgG) in various immunoassays and immunochemical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ecl detection kit, by GE Healthcare (2 mentions)
Versadoc system, by Bio-Rad (2 mentions)
Pvdf transfer membrane, by Merck Group (2 mentions)
Horseradish peroxidase hrp conjugated donkey anti rabbit igg, by GE Healthcare (1 mentions)
Sc 7196, by Santa Cruz Biotechnology (1 mentions)
Mab374, by Merck Group (1 mentions)
Hrp conjugated donkey anti rabbit igg, by GE Healthcare (1 mentions)
Carnosine, by Fujifilm (1 mentions)
Np 40 substitute, by Fujifilm (1 mentions)
Nupage novex 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Iblot transfer stack, by Thermo Fisher Scientific (1 mentions)
Nicl2, by Fujifilm (1 mentions)
Zncl2, by Fujifilm (1 mentions)
Ripa buffer, by Fujifilm (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
6 ohda, by R&D Systems (1 mentions)
Celltiter glo 2, by Promega (1 mentions)
Thunderbird next sybr qpcr mix, by Toyobo (1 mentions)
Antibody against actin, by Santa Cruz Biotechnology (1 mentions)
Dulbecco s modified eagle s medium ham s nutrient mixture f 12 dmem ham s f12, by Fujifilm (1 mentions)
4 protocols using hrp conjugated donkey anti mouse igg
1
Western Blot Analysis of PPAR-γ
2
Western Blot Analysis of Cytoplasmic Proteins
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Cytoplasmic protein (50 μg) was dissolved in Laemmli's buffer, boiled for 10 min, electrophoresed on a 10% SDS polyacrylamide gel, and electroblotted onto a PVDF transfer membrane (Millipore, Billerica, MA, USA). The membrane was blocked with PBS containing 5% skim milk and 0.05% Tween 20 and then incubated for 1 h with each antibody (diluted to 1:500 with blocking buffer). After washing, the membrane was incubated with 1:5000 diluted horseradish peroxidase (HRP)-conjugated Donkey anti-Rabbit IgG or HRP-conjugated Donkey anti-Mouse IgG (GE Healthcare, Little Chalfont, UK) in Blocking Buffer. Subsequently, the blots were developed using the ECL Detection Kit (GE Healthcare) and protein bands were visualized using the VersaDoc System (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
3
Investigating Zinc-Mediated CHOP Regulation
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An antibody against actin (SC-47778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against CHOP (#5554) and goat anti-rabbit IgG (horseradish peroxidase- (HRP-) conjugated, #7074) were purchased from Cell Signaling Technology Japan (Tokyo, Japan). Tauroursodeoxycholic acid (TUDCA) was purchased from Tokyo Chemical Industry (Tokyo, Japan). The Zinc Assay Kit was purchased from Metallogenics Co. Ltd. (Chiba, Japan). HRP-conjugated donkey anti-mouse IgG was purchased from GE Healthcare Japan (Tokyo, Japan). Carnosine, ZnCl2, NiCl2, and RIPA buffer (20 mmol/L Tris-HCl (pH 7.4), 0.05 w/v% NP-40 substitute, 2.5 mmol/L MgCl2, and 200 mmol/L NaCl) were purchased from Wako Pure Chemicals (Tokyo, Japan). Protease and phosphatase inhibitors (87786 and 78420), NuPAGE® Novex 4–12% Bis-Tris Protein Gel, iBlot™ Transfer Stack, and SuperSignal™ West Dura Extended Duration Substrate were from Thermo Fisher Scientific K.K. (Tokyo, Japan).
4
Quantitative Analysis of Oxidative Stress in Cell Lines
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6-OHDA was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). CellTiter-Glo® 2.0 was purchased from Promega Corporation (Madison, WI, USA). Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/Ham’s-F12) and low-glucose DMEM were purchased from Fujifilm Wako Pure Chemical Corporation (Tokyo, Japan). FastGene™ RNA Basic kit was obtained from Nippon Genetics Co., Ltd. (Tokyo, Japan), PrimeScript™ RT master mix (Perfect Real Time) was obtained from Takara Bio (Shiga, Japan), and THUNDERBIRD® Next SYBR® qPCR mix was obtained from Toyobo (Osaka, Japan). 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) was obtained from Merck KGaA (Darmstadt, Germany), and MitoSOX™ was obtained from Thermo Fisher Scientific (Waltham, MA). Antibody against actin (Code: SC-47778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phospho-JNK (p46 and p54) (Code: #4668), phospho-p38 (Code: #4511), phospho-ERK (Code: #4376), and IκBα (Code: #4814) and goat anti-rabbit IgG, HRP-linked antibody (Code: #7074) were purchased from Cell Signaling Technology Japan (Tokyo, Japan). HRP-conjugated donkey anti-mouse IgG was purchased from GE Healthcare (Tokyo, Japan).
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