HFFF-TERTs were used for all experiments apart from Figure 6 C, where 293T cells were used. For most immunoblots, cells were lysed with RIPA buffer (Cell Signaling) containing Complete Protease Inhibitor Cocktail (Roche) and then lysates were sonicated. For cells infected by single gene deletion viruses, 6 M Guanidine whole cell lysates were precipitated using a ProteoExtract protein precipitation kit (Calbiochem) and re-dissolved in 2% SDS/Tris 200 mM pH 8.5 with sonication. Protein concentration was measured by BCA (Pierce). Lysates were reduced with 6X Protein Loading Dye (Tris 375 mM pH 6.8, 12% SDS, 30% glycerol, 0.6 M DTT, 0.06% bromophenol blue) for 5 min at 95°C. 50 μg of protein for each sample was separated by PAGE using 4-15% TGX Precast Protein Gels (Bio-rad), then transferred to PVDF membranes using Trans-Blot Systems (Bio-rad). The following primary antibodies were used: anti-HLTF (ab17984, Abcam), anti-HCMV IE1/2 (ab53495, Abcam), anti-GAPDH (MAB5718, R&D Systems), anti-V5 (MA5-15253, Thermo), anti-Sp100 (GTX131570, GeneTex). Secondary antibodies were IRDye 680RD goat anti-mouse (925-68070, LI-COR) and IRDye 800CW goat anti-rabbit (925-32211, LI-COR). Fluorescent signals were detected using a LI-COR Odyssey, and images were processed using Image Studio Lite (LI-COR).
Mab5718
Manufactured by R&D Systems
Sourced in United States
MAB5718 is a mouse monoclonal antibody that targets the human Haptoglobin protein. This antibody is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor cocktail, by Roche (2 mentions)
Ab17984, by Abcam (1 mentions)
Ab53495, by Abcam (1 mentions)
Ma5 15253, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw goat anti rabbit, by LI COR (1 mentions)
Image studio lite, by LI COR (1 mentions)
Anti sp100, by GeneTex (1 mentions)
Irdye 680rd goat anti mouse, by LI COR (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Beyoecl plus detection system, by Beyotime (1 mentions)
Anti gapdh, by R&D Systems (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Af1065, by R&D Systems (1 mentions)
Ab32249, by Abcam (1 mentions)
Ab27794, by Abcam (1 mentions)
Mab971, by Abcam (1 mentions)
Ab16051, by R&D Systems (1 mentions)
Ba1034, by Thermo Fisher Scientific (1 mentions)
Nl004, by R&D Systems (1 mentions)
Ab38898, by Abcam (1 mentions)
5 protocols using mab5718
1
Immunoblotting of HLTF and HCMV Proteins
2
Western Blot Analysis of Protein Expression
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The cells and tumor tissues were collected and lysed on ice. Subsequent to centrifugation at 12,000 × g for 20 min, the concentration of proteins was measured and protein samples were denatured by boiling for 10 min, then were and loaded onto a 10% SDS-PAGE gel for electrophoresis. The proteins were transferred onto a PVDF membrane (EMD Millipore, Billerica, MA, USA) which was then incubated in the blocking solution at room temperature for 2 h. Anti-C14orf166 (1:100; catalog no. 19848-1-AP; ProteinTech Group, Inc., Chicago, IL, USA) and anti-GAPDH (1:5,000; catalog no. MAB5718; R&D Systems, Inc., Minneapolis, MN, USA) were used for western blotting, incubated at 4°C overnight. The membranes were subsequently incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:6,000; catalog no. NB730-H; Novus Biologicals, LLC, Littleton, CO, USA) or HRP-labeled goat anti-mouse IgG (1:1,000; catalog no. HAF007; Novus Biologicals, LLC), for 1.5 h at room temperature. Protein expression was normalized against GAPDH expression. Bands were visualized with the BeyoECL Plus Detection System (Beyotime Institute of Biotechnology, Haimen, China) and Bio-Rad ImageLab software version 3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
3
Lysis and Antibody Detection
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Cell lines 3T3-L1 and 3T3-F442A were disrupted by hypotonic lysis in sterile water. Human CRP antibody AF1707, human TNF-α antibody MAB210-100, human adiponectin/Acrp30 antibody (AF1065), human resistin antibody MAB13591-100, and human GAPDH antibody MAB5718 were from R&D Systems (Minneapolis, MN, USA).
4
Protein Expression Analysis in Trophoblast Cells
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The total proteins of B6Tert, HTR8/SVneo, and JEG-3 cells were collected and cracked by high RIPA buffer (Solarbio, Beijing, China). The concentration of proteins was detected using a BCA kit (Yeasen, Shanghai, China). Then, the SDS-PAGE was used to separate the proteins, and the proteins were transferred to a nitrocellulose (NC) membrane (Haoran, Shanghai, China). The NC membrane was blocked by 5% non-fat milk at room temperature for 1.5 h, and then incubated with the primary antibodies (anti-BRIT1, Abcam, ab121277, dilution: 1: 900; anti-matrix metalloproteinase-2 (MMP-2), R&D, IC903G, dilution: 1: 700; anti-MMP-9, Abcam, ab38898, dilution: 1: 1000; anti-tissue inhibitor of metalloproteinases-1 (TIMP-1), R&D, IC970G, dilution: 1: 700; anti-TIMP-2, R&D, MAB971, dilution: 1: 500; anti-Wnt2, Abcam, ab27794, dilution: 1: 600; anti-Wnt3, Abcam, ab32249, dilution: 1: 600; anti-β-catenin, Abcam, ab16051, dilution: 1: 600; anti-GAPDH, R&D, MAB5718, dilution: 1: 800) at 4°C overnight. Subsequently, the NC membrane was incubated with the secondary antibodies at room temperature for 1.5 h (goat anti-mouse IgG, Abcam, ab6785, 1: 8000; donkey anti-rabbit IgG, R&D, NL004, 1: 5000; mouse anti-rabbit IgG, Invitrogen, BA1034, 1: 7000). Chemiluminescence detection was carried out using ECL reagent (Huiying, Shanghai, China).
5
Quantifying HIF-1α in Kidney Tissue
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HIF-1α was measured in kidney tissue by a Western blot methodology. Briefly, pools of 100 mg of kidney tissue were mechanically homogenized in 500 µL of 1X lysis buffer containing 10 mM Tris-HCl (pH 7.5); 50 mM KCl, 2 mM MgCl2, 1% Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and cOmplete™ Protease Inhibitor Cocktail according to manufacturer conditions (Roche, Basilea, Switzerland) at 4 °C and centrifuged at 13,000 g for 5 min. Proteins were quantified in the supernatant by the Bradford method (Bio-Rad, Hercules, CA, USA) and 150 µg of protein was separated on 12% polyacrylamide gels. Proteins were transferred onto PVDF membranes and monoclonal mouse antibodies were used for the detection of HIF-1α (sc-13515; 1:300, 6.7 µg/mL, Santa Cruz Biotechnology, Dallas, TX, USA) and GAPDH (MAB5718; 1:10,000; 0.05 µg/mL; R&D Systems, Minneapolis, MN, USA). A secondary HRP-conjugated anti-mouse antibody was used (W4021; 1:10,000; 0.1 µg/mL; Promega, Madison, WI, USA), and the signal was measured by chemiluminescence using a Clarity™ Western ECL Substrate (Bio-Rad).
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