Proteins were extracted from the same samples harvested for viral RNA quantification, above. Extractions from buffer RLT were performed using the iced acetone method described by the manufacturer (Qiagen). Proteins were separated by denaturing SDS polyacrylamide gel electrophoresis, and transferred to PVDF (Pall Corp., Pensacola, FL). Immunoblotting was performed with monoclonal antbody to influenza NP (AA5H; AbCam, Cambridge, MA) or anti-M1 polyclonal (a kind gift of Dr. Adolfo García-Sastre, Icahn School of Medicine at Mount Sinai), and peroxidase conjugated secondary antiserum. Blots were imaged with Supersignal substrate (ThermoFisher Scientific, Carlsbad, CA), on a Cell Biosciences FluorChem HD2. Consistent loading was monitored by Coomassie Brilliant Blue R-250 (Amresco, Solon, OH) staining of the post-transfer gel.
Fluorchem hd2
Manufactured by Cell Biosciences
Sourced in United Kingdom, Canada
The FluorChem HD2 is a high-performance imaging system designed for the detection and quantification of fluorescent and chemiluminescent signals. It features a sensitive CCD camera, various illumination options, and advanced software for image acquisition and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Coomassie brilliant blue r 250, by Avantor (1 mentions)
Polyvinylidene fluoride (pvdf), by Pall Corporation (1 mentions)
Ecl select western blotting detection reagent, by GE Healthcare (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Foxm1, by Cell Signaling Technology (1 mentions)
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
Amersham ecl prime solution, by GE Healthcare (1 mentions)
Pro prep lysis buffer, by iNtRON Biotechnology (1 mentions)
Supersignal west dura chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Gtx121937, by GeneTex (1 mentions)
Ecl prime solution, by GE Healthcare (1 mentions)
Goat anti rabbit igg secondary antibody, by Jackson ImmunoResearch (1 mentions)
8 protocols using fluorchem hd2
1
Influenza Protein Extraction and Immunoblotting
2
Ibrutinib-Induced BCR Signaling and Apoptosis Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lines treated or non-treated with ibrutinib were harvested and lysed using RIPA buffer (Cat No. 9806, Cell Signaling Technology, Danvers, MA, USA) supplemented with protease inhibitor cocktail tablets (Cat No. 04693124001, Roche, Mannheim, Baden-Wuerttemberg, Germany) to get the protein in total cell extracts. Then equivalent amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on which separating gel contained 4-15% acrylamide (Cat No. 456–1083, Bio-Rad, Hercules, CA, USA), transferred to polyvinylidene fluoride (PVDF) membrane (Cat No. IPVH00010, Millipore Corporation, Billerica, MA, USA) and probed with appropriate primary and secondary antibodies mentioned above to study the BCR signaling pathway and apoptosis-related protein. ECL select western blotting detection reagent (Cat No. RPN 2235, GE Healthcare, Little Chalfront, Bukinghamshire, UK) was used for detection on a Fluor Chem HD2 (Cell Biosciences, Santa Clara, CA, USA).
3
AMPK and FOXM1 Signaling in PDAC Cells
4
Quantitative Western Blot Analysis
5
Quantitative HOXA11 Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The whole protein extracts from the tissue samples were prepared using a PRO-PREP lysis buffer (#17081, iNtRon Biotechnology, Seongnam, Korea), and the protein concentrations were determined using a bicinchoninic acid protein assay (#23227, Thermo Fisher Scientific, Waltham, MA). The proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by blotting onto nitrocellulose membranes, and probing with the antibodies against HOXA11 (#SC-48542, 1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA). The blotted membranes were then incubated with the goat anti-rabbit IgG secondary antibody for 1 hour. Subsequently, the membranes were incubated in an Amersham ECL-prime solution (#RPN2232, GE Healthcare Life Sciences, Pittsburgh, PA) in the dark for 1 minute and exposed under FluorChemHD2 (Cell Biosciences, Santa Clara, CA) for visualization. Only the samples with a consistent result from repeated experiments were selected for analysis. The densities of the bands were measured using free image analyzer software (ImageJ V1.8x, National Institutes of Health, http://rsb.info.nih.gov/ij/ ). The results are presented as the mean±standard error of mean calculated from independent samples.
6
Western Blot Quantification Workflow
7
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For protein isolation, cells were sonicated in 100 µl cold 1% sodium dodecyl sulphate (SDS) containing protease inhibitor (Roche, Basel, Switzerland). Western blots were performed as previously described [67 (link)], and the membranes were incubated with primary antibodies overnight at 4°C (S6 ). Chemiluminescence was detected using a FluorChem HD2 camera with the FluorChem software (Cell Biosciences, Santa Clara, CA). Band intensities were quantified using NIH Image J64 software and normalized relative to GAPDH.
8
Quantitative Analysis of AKT and PTEN Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole protein extracts of the cells for western blotting were prepared using PRO-PREP lysis buffer (Intron, #17081), and protein concentrations were determined using the BCA protein assay (Thermo Fisher Scientific, #23227). Proteins were separated by 10% SDS-PAGE, blotted onto nitrocellulose membranes, and then probed with antibodies against total AKT (Genetex, #GTX121937, 1:3000 dilution), phosphorylated AKT (Genetex #GTX61708, 1:2000 dilution), total PTEN (Genetex, #GTX101025, 1:500 dilution), and phosphorylated PTEN (Genetex, #GTX61780, 1:1000 dilution). The membranes were then incubated with a goat anti-rabbit IgG secondary antibody (Jackson, #003318367, 1:4000 dilution) for 1 hour. The membranes were incubated in ECL-prime solution (GE Healthcare Amersham, #RPN2232) in the dark for 1 minute and then exposed under a fluorchemHD2 (Cell biosciences) for visualization.
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!