Cells were homogenized in a RIPA buffer supplemented with protease and phosphatase inhibitor cocktails (Roche). The homogenates were centrifuged at 13,000 r.p.m for 10 minutes, and the supernatants were used as whole-cell lysates. Protein concentration was assessed by Bio-Rad BCA protein assay. 30 ug of protein lysate was separated by SDS-PAGE and then transferred on to a PVDF membrane. The membranes were probed with primary anti-CDC6 (Bethyl Laboratories Inc., cat. A302-487A-T-1), anti-NCBP2 (Bethyl Laboratories Inc., cat. A302-553A-T), anti-SHMT2 (Cell Signaling, cat. 12762), anti-MTHFD2 (Cell Signaling, cat. 41377) – all at 1:1,000 dilution – or anti-actin (Santa Cruz Biotechnologies) – at 1:500 dilution – for 1 h. Membranes were washed and then incubated with HRP-conjugated donkey anti-rabbit (Santa Cruz Biotechnologies) at 1:5,000 dilution or HRP-conjugated donkey anti-goat (Santa Cruz Biotechnologies) at 1:10,000 dilution. Protein was detected with Western Lightning Plus-ECL (Perkin Elmer), and analyzed using ImageJ Software ver. 1.50 (NIH, USA).
Hrp conjugated donkey anti goat
Manufactured by Santa Cruz Biotechnology
Sourced in Canada, United Kingdom
The HRP-conjugated donkey anti-goat is a secondary antibody that is used to detect the presence of goat primary antibodies in various immunoassay techniques. The horseradish peroxidase (HRP) enzyme conjugated to the antibody facilitates the detection of the target antigen through a colorimetric or chemiluminescent reaction.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated donkey anti rabbit, by Santa Cruz Biotechnology (2 mentions)
Anti actin, by Santa Cruz Biotechnology (2 mentions)
Anti shmt2, by Cell Signaling Technology (1 mentions)
Bca protein assay, by Bio-Rad (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Mouse anti α tubulin antibody, by Merck Group (1 mentions)
Mouse anti e cadherin, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Hrp conjugated goat anti mouse, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Versadoc 5000 mp imaging system, by Bio-Rad (1 mentions)
Anti mcl 1, by Rockland Immunochemicals (1 mentions)
Quantity one 1 d, by Bio-Rad (1 mentions)
Mouse monoclonal anti actin, by Merck Group (1 mentions)
Molecular imager chemidoc xrs system, by Bio-Rad (1 mentions)
Irdye 680cw goat anti mouse, by LI COR (1 mentions)
Irdye 800cw goat anti rabbit, by LI COR (1 mentions)
Mouse monoclonal anti chk1, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg, by LI COR (1 mentions)
7 protocols using hrp conjugated donkey anti goat
1
Protein Extraction and Immunoblotting
2
Western Blot Antibody Reagents
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Anti-mouse α-tubulin antibody was from Sigma-Aldrich, Inc., St Louis, MO. Rat monoclonal anti-Snail and HRP-conjugated goat anti-rat antibodies were from Cell Signaling Technology, Inc., Danvers, MA. Goat monoclonal anti-Cat L and goat monoclonal anti-vimentin antibodies were purchased from R&D Systems (Minneapolis, MN). The HRP conjugated donkey anti-goat, anti-mouse Twist, and anti-mouse E-cadherin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated sheep anti-mouse and sheep anti-goat were purchased from Amersham Biosciences, Buckinghamshire, UK. Luminata Forte The anti-mouse Occludin antibody was purchased from Invitrogen (Carlsbad, CA). HRP chemiluminescence detection reagent was purchased from EMD Millipore (Billerica, MA). The protease inhibitor cocktail was from Roche Molecular Biochemicals, Indianapolis, IN.
3
Detecting Viral Env Proteins via IFA
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Indirect immunofluorescence assays (IFAs) were performed on the 293T and DF1 cells. The monoclonal antibody JE9, which is specific to the Env of ALV-J, was used as the primary antibody19 (link). FITC-goat anti-mouse IgG was used as the secondary antibody. Western blot analyses were performed on cell lysates. JE9 or anti-β-actin or anti-chANXA2 antibody was used as the primary antibody, and HRP-conjugated goat anti-mouse or HRP-conjugated donkey anti-goat was used as the secondary antibody (Santa Cruz).
4
Western Blot Analysis of Mcl-1 Protein
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Two hundred GV oocytes from approximately 8–10 Mcl-1cKO and Mcl-1+/+ females were collected and lysed in RIPA buffer. Lysates were separated by SDS-PAGE and then transferred on to a PVDF membrane. Blots were incubated with anti-Mcl-1 (Rockland Immunochemicals, 600-401-394S) or anti-actin (Santa Cruz Biotechnologies, sc-1616). Membranes were washed and then incubated with specific HRP-conjugated donkey anti-rabbit (Santa Cruz Biotechnologies) or HRP-conjugated donkey anti-goat (Santa Cruz Biotechnologies) and detected using enhanced chemiluminescence (Thermo Scientific, Burlington, ON, Canada) SuperSignal West Femto Chemiluminescent Substrate and then imaged on the VersaDoc 5000MP Imaging System (Bio-Rad, Mississauga, ON, Canada).
5
Western Blot Analysis of UCP2 in Mouse Islets
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Protein extracts from mouse islets treated as described were subjected to electrophoresis on a 12% polyacrylamide gel, electro-transferred onto nitrocellulose membrane, and blocked with 3% BSA in PBS (1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM NaCl and 2.7 mM KCl at pH 7.3). Membranes were then incubated overnight at 4 °C with different antibodies: goat anti-human polyclonal antibody to UCP2 (1:1000, #6527, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal anti-actin (1:5000, #4700, Sigma-Aldrich) in PBS containing 3% BSA and 0.05% Tween-20. After washing 3 times with PBS supplemented with 0.05% Tween-20, membranes were incubated with a horseradish peroxidase (HRP)-conjugated donkey anti-goat (1:10000, #2056, Santa Cruz) or anti-mouse antibody (1:5000, NA931, Amersham Biosciences, UK) for 1hr at room temperature. After washes, the immunoreactivity was visualized by SuperSignal West Pico Chemiluminescent Substrate system (Pierce Biotechnology, Inc., Rockford, IL) and Molecular Imager ChemiDoc XRS system (Bio-Rad, Hercules, CA) controlled by Quantity One 1-D (Bio-Rad) analysis software.
6
Cell Lysis and Western Blot Analysis
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Whole-cell lysates were prepared as follows: cells were harvested, resuspended in commercial RIPA buffer (Beyotime, P0013B), and then subjected to sonication followed by centrifugation. Western blotting was carried out as previously described [14 (link)]. The primary antibodies used for western blotting were as follows: mouse monoclonal anti-GAPDH (Abmart, 3B3), rabbit polyclonal anti-POT1 (Novus, NB500-176), goat polyclonal anti-ATR (Santa Cruz Biotechnology, sc-1887), rabbit polyclonal anti-phospho-ATR (Abcam, ab178407), mouse monoclonal anti-Chk1 (Santa Cruz Biotechnology, sc-377231), rabbit monoclonal anti-phospho-Chk1 (Cell Signaling Technology, No.2348L). The secondary antibodies used were HRP-conjugated donkey-anti-goat (Santa Cruz Biotechnology, sc2020), fluorescein-conjugated IRDye-680CW goat anti-mouse (LI-COR, 926-32220) and IRDye-800CW goat anti-rabbit (LI-COR, 926-32211).
7
Antibody Characterization for Western Blotting
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The rabbit polyclonal anti-GRP170 (1:1000 for western blotting), and mouse monoclonal anti-ERdj4 (0.8ug/ml for western blotting) antibodies were produced in the Hendershot lab. The mouse monoclonal anti-HA antibody was a kind gift from Dr. Al Reynolds (Vanderbilt University, 1:500 for IP, 1:1000 for western blotting). Other antibodies used were obtained commercially and include: goat anti-mouse λ LC (SouthernBiotech, 1060-01, 1:500 for IP,1:1000 for western blotting), HRP-conjugated goat anti-rabbit (Santa Cruz Biotechnology, sc-2054, 1:10,000), HRP-conjugated donkey anti-goat (Santa Cruz Biotechnology, sc-2020, 1:10,000), HRP-conjugated goat anti-mouse (SouthernBiotech, 1038-05, 1:10,000), monoclonal anti-ERdj5 (Abnova, H00054431-M01, 1:1,000 for western blotting), polyclonal rabbit anti-pro-SP-C (Seven Hills Bioreagents, WRAB9337, rabbit bleed 364, 1:2,000 for IP, 1:5000 for western blotting), mouse monoclonal anti-β-Actin (Sigma-Aldrich, AC-15, 1:2,000 for western blotting), and IRdye® secondary antibodies (LI-COR Biosciences, all 1:20,000): goat anti-mouse IgG (925-32210), goat anti-rabbit IgG (925-68071), and donkey anti-goat IgG (926-68074).
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