Total protein was extracted from cell cultures and electrophoretically separated and blotted as described previously.54 (link) For antigen detection, rabbit anti-K5 (AF138, Covance), 1:2,000, was used, and Coomassie staining was used to confirm equal loading. Horseradish peroxidase-conjugated species-specific secondary antibodies (Dianova) were used, and immunodetection was performed using Super Signal West Pico and Dura substrates (Pierce, Thermo Scientific). Densitometry ratios of each sample were calculated and presented as the fraction of change from the control expression value. The densitometry ratios from each sample were averaged (n = 3), and the data are presented as mean value ± SEM.
Horseradish peroxidase conjugated species specific secondary antibodies
Manufactured by Dianova
Horseradish peroxidase-conjugated species-specific secondary antibodies are laboratory reagents used in various immunoassay techniques. They consist of secondary antibodies that are chemically conjugated to the enzyme horseradish peroxidase. These antibody-enzyme conjugates are designed to detect and quantify specific target proteins or molecules in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti gst, by GE Healthcare (2 mentions)
Tween 20, by Thermo Fisher Scientific (2 mentions)
Anti flag, by Merck Group (2 mentions)
Las 4000 imaging system, by Bio-Rad (2 mentions)
Western lightning chemiluminescence reagent plus, by PerkinElmer (2 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Chemiluminescence reagent plus, by PerkinElmer (1 mentions)
Anti gfp, by Cell Signaling Technology (1 mentions)
Anti v5, by Thermo Fisher Scientific (1 mentions)
Anti ha ha 11 clone 16b12, by Fortrea (1 mentions)
Odyssey sa imaging system, by LI COR (1 mentions)
4 protocols using horseradish peroxidase conjugated species specific secondary antibodies
1
Quantifying Protein Expression Changes
2
Western Blot Analysis of Viral Proteins
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Proteins were separated in polyacrylamide-Tricine gels. After SDS-PAGE, proteins were transferred onto a nitrocellulose membrane (Pall, USA). The membrane was blocked with 5% (w/v) dried skim milk in phosphate-buffered saline with 0.05% (v/v) Tween 20 (Invitrogen). For antigen detection, mouse monoclonal antibody against NS3 of the JFH-1 isolate (4D11; [13 (link)]) or anti-NS3 (2E3) [37 (link)], anti-FLAG (Sigma), anti-GFP (Cell signalling) and anti-GST (GE Healthcare), antibodies were used in 2% (w/v) dried skim milk in phosphate-buffered saline with 0.05% (v/v) Tween 20. For primary antibody detection, horseradish peroxidase-conjugated species-specific secondary antibodies (Dianova) were used at a 1:3000 dilution and Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer) was applied prior to imaging using a LI-COR imaging system (Biorad, Munich).
3
Western Blot Analysis of HCV Proteins
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Proteins were separated in polyacrylamide-Tricine gels. After SDS-PAGE, proteins were transferred onto a nitrocellulose membrane (Pall, USA). The membrane was blocked with 5% (w/v) dried skim milk in phosphate-buffered saline with 0.05% (v/v) Tween 20 (Invitrogen). For antigen detection, anti-NS5A 9E10 [60 (link)], mouse monoclonal antibody against NS3 of the JFH-1 isolate (4D11) (generated in a cooperation between Harish Ramanathan, Michael Engle, Michael S. Diamond and Brett D. Lindenbach) or anti-NS3 (2E3) [61 (link)], anti-NS2 (YAL-4-70-8, Cell Essentials), anti-NS4B [62 (link)], anti-FLAG (Sigma), anti-V5 (Invitrogen), anti-HA (HA.11 clone 16B12, Covance) and anti-GST (GE Healthcare), antibodies were used in 2% (w/v) dried skim milk in phosphate-buffered saline with 0.05% (v/v) Tween 20. For primary antibody detection, horseradish peroxidase-conjugated species-specific secondary antibodies (Dianova) were used at a 1:3000 dilution and Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer) was applied prior to imaging using a LAS 4000 imaging system (Biorad, Munich). Quantifications of Western blots were carried out using ImageJ 1.47t software (NIH, Bethesda).
4
Quantification of Viral Polyprotein Cleavage
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Quantifications of NS2 cleavage efficiencies were carried out using ImageJ 1.47t (NIH, Bethesda). The quantification of cleavage efficiencies was done by the determination of Western blot signal intensities for uncleaved polyprotein precursor and cleaved product to calculate the NS2 cleavage rate. The respective primary antibodies were either detected by IRDye 800CW-labeled secondary antibodies using the Odyssey SA imaging system (LI-COR) or by horseradish peroxidase-conjugated species-specific secondary antibodies (Dianova) using the LAS 4000 imaging system (Biorad, Munich) and Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer).
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