Cells were lysed in NP-40 lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% NP-40, Halt Protease Inhibitor). Lysates were quantified by Bradford assay (Carl Roth). In general, 30 μg per sample were separated by SDS-PAGE, transferred onto PVDF membranes and probed with different primary antibodies. Endogenous SAMHD1 was probed with anti-SAMHD1 (3F5) antibody (novusbio); phosphorylated T592 was detected with a pT592-SAMHD1-specific antibody (ProSci). Myc-tagged proteins were probed with an anti-myc (9B11) antibody (Cell Signaling), FLAG-tagged proteins with anti-FLAG M2 antibody (Sigma), T7-tagged proteins with anti-T7 antibodies (Novagen, Abcam), and HA-tagged proteins with an anti-HA (16B12) antibody (Biolegend). To control for equal loading of cell lysates membranes were probed with anti-HSP90 α/β antibody (Santa Cruz). Subsequently, PVDF membranes were incubated with anti-mouse or anti-rabbit HRP-labeled secondary antibodies (Cell Signaling) and visualized using HRP substrate on an Intas Advanced Fluorescence Imager (Intas).
Anti mouse or anti rabbit hrp labeled secondary antibodies
Manufactured by Cell Signaling Technology
Anti-mouse or anti-rabbit HRP-labeled secondary antibodies are laboratory reagents used to detect and visualize target proteins in Western blotting, immunohistochemistry, and other immunoassays. These antibodies specifically bind to the constant regions of primary antibodies raised against mouse or rabbit antigens, and the conjugated horseradish peroxidase (HRP) enzyme allows for colorimetric or chemiluminescent detection of the target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bradford assay, by Carl Roth (2 mentions)
Anti ha antibody 16b12, by BioLegend (1 mentions)
Anti myc antibody 9b11, by Cell Signaling Technology (1 mentions)
Anti hsp90 α β antibody, by Santa Cruz Biotechnology (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
Advanced fluorescence imager, by Intas (1 mentions)
Hsp90 α β, by Santa Cruz Biotechnology (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
2 protocols using anti mouse or anti rabbit hrp labeled secondary antibodies
1
Western Blot Analysis of SAMHD1 Phosphorylation
2
Immunoblot Analysis of SARS-CoV-2 Proteins
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Cells were lysed in NP‐40 lysis buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% NP‐40, Halt Protease Inhibitor). Protein content of the lysates was determined by Bradford assay (Carl Roth). Samples were separated by SDS–PAGE, transferred onto Immobilon‐P PVDF membranes (Merck), and probed with primary antibodies targeting endogenous IFIT‐1 (Cell Signaling, 14769), SARS–CoV2 Nucleocapsid protein (novubio, NB100‐56576), and the house keeping genes HSP90 α/β (Santa Cruz, sc‐13119) or GAPDH (Cell Signaling, 2118). Membranes were probed with anti‐mouse or anti‐rabbit HRP‐labeled secondary antibodies (Cell Signaling).
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