HEK293 cells pre-plated in a 6-well plate were transiently transfected with 2.5 μg DNA vaccine plasmids with Hieff TransTM Liposomal Transfection Reagent (YEASEN, Shanghai, China). Two days later, the cells were pelleted and lysed in lysis buffer. Cell lysates were separated by SDS-PAGE and transferred to PVDF membranes. Immunoblotting was performed by using rabbit anti-RBDWT primary Ab (Bioworld, Nanjing, China) diluted 1:1,000 in 5% milk–0.05% PBS-Tween 20, and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary Ab (BD Biosciences, San Diego, CA, USA). Chemiluminescence detection was performed with the ECL Prime Western Blotting System and acquired by the ChemiDoc Imaging System (Bio-Rad).
Horseradish peroxidase
Manufactured by BD
Sourced in China, United States
Horseradish peroxidase is an enzyme derived from the horseradish plant. It is commonly used as a labeling agent in various immunoassays, blotting techniques, and other biochemical applications due to its ability to catalyze the oxidation of substrates in the presence of hydrogen peroxide.
Automatically generated - may contain errors
Lab products found in correlation
Hieff trans liposomal transfection reagent, by Yeasen (2 mentions)
Rabbit anti rbdwt primary ab, by Bioworld Technology (2 mentions)
Hrp labeled goat anti rabbit igg secondary ab, by BD (2 mentions)
Ecl prime western blotting system, by Bio-Rad (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Genesnap software, by Syngene (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Western lightning western blot chemiluminescence reagent, by PerkinElmer (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase, by Cell Signaling Technology (1 mentions)
Ab14041, by Abcam (1 mentions)
Ab23981, by Abcam (1 mentions)
Ab205394, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (1 mentions)
6 protocols using horseradish peroxidase
1
Quantifying SARS-CoV-2 RBD Expression
2
RBD Protein Expression in HEK293 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293 cells pre-plated in a 6-well plate were transiently transfected with 2.5 μg DNA vaccine plasmids with Hieff TransTM Liposomal Transfection Reagent (YEASEN, Shanghai, China). Two days later, the cells were pelleted and lysed in immunoprecipitation assay buffer. Cell lysates and supernatants were separated by SDS-PAGE and transferred to PVDF membranes. Immunoblotting was performed by using rabbit anti-RBDWT primary Ab (Bioworld, Nanjing, China) diluted 1:1000 in 5% milk-0.05% PBS-Tween 20 and horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary Ab (BD Biosciences, San Diego, CA, USA). Chemiluminescence detection was performed with the ECL Prime Western Blotting System and acquired by the ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA).
3
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were isolated from tissues and cells using RIPA lysis buffer (Thermo Fisher) and quantified using a BCA analysis kit (BioVision, USA). The proteins were separated on a 10 % SDS-polyacrylamide gel and transferred to a PVDF membrane (Thermo Fisher) which was covered with 5 % milk and incubated with the primary antibody overnight and the secondary antibody conjugated with horseradish peroxidase (BD Biosciences) for 1h. Protein bands were evaluated using the SynGene system and GeneSnap software (SynGene, USA). Primary antibodies: N-cadherin (13116, Cell Signaling Technology), GAPDH (2118, Cell Signaling Technology), E-cadherin (3195, Cell Signaling Technology), cleaved caspase-3 (9664, Cell Signaling Technology), Ki-67 (ab16667, Abcam).
4
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NSCs were washed with phosphate-buffered saline (PBS), lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM NaF, 1 mM Na3VO4, 1% NP-40, 0.5% sodium deoxycholate, and 1% sodium dodecyl sulfate, pH 7.5, supplemented with a complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN), and then centrifuged at 12,000 × g and 4℃ for 10 min. Supernatants (cell lysates) were collected, and the protein concentrations were measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (8% gel) under reducing conditions and transferred to polyvinylidene difluoride membranes. The membranes were probed with primary antibodies followed by appropriate secondary antibodies conjugated with horseradish peroxidase (BD Biosciences). Signals were visualized with Western Lightning Western blot chemiluminescence reagent (Perkin Elmer Life and Analytical Sciences, Boston, MA). The bands were quantified using the NIH ImageJ. Densitometric values were normalized by setting the NICD/actin or EGFR/actin protein ratio for control (WT) in each treatment. The normalized value from control (WT) is defined as 1.0.
5
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were isolated from cells using radioimmunoprecipitation assay lysis bu er ( ermo Fisher) and quanti ed using a bicinchoninic acid kit (BioVision, USA). e proteins were separated on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene uoride membrane (Thermo Fisher). The membrane was incubated in 5% milk and then with the primary antibody overnight and with the secondary antibody conjugated with horseradish peroxidase (BD Biosciences) at room temperature for 1 h. Protein bands were evaluated using the SynGene system and GeneSnap so ware (SynGene, USA). Primary antibodies: glyceraldehyde-3-phosphate dehydrogenase (2118, Cell Signaling Technology), cleaved caspase-3 (9664, Cell Signaling Technology), cleaved caspase-9, and cytochrome c (Cyto C) (37BA11, Invitrogen).
6
Protein Isolation and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteins were isolated using radioimmunoprecipitation assay lysis buffer (Thermo Fisher) and quantified using the BCA Kit (BioVision, USA). After separation by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and loading onto polyvinylidene fluoride membranes (Thermo Fisher), the proteins were blocked with 5% milk and incubated with primary antibodies overnight and a secondary antibody conjugated with horseradish peroxidase (BD Biosciences) at room temperature for 1 h. Protein banding was evaluated using the SynGene system and GeneSnap software (SynGene, USA). Primary antibodies: Glyceraldehyde-3-phosphate dehydrogenase (2118, Cell Signaling Technology), Periostin (ab14041, Abcam), BMP1 (ab205394, Abcam), osteopontin (OPN) (AB5405, MilliporeSigma), runt-related transcription factor 2 (RUNX2) (ab23981, Abcam).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!