Total protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (including protease inhibitor cocktail) [15 (link)]. After measuring the protein concentration using a BCA assay kit (Thermo Scientific, MA, USA), the protein was subjected to Western blotting. Equal amounts of protein from each sample were separated using 8%, 15% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk at room temperature (22–25 °C) for 1 h and then incubated with a monoclonal rabbit anti-α-Syn antibody (1:10000, Abcam), a polyclonal mouse anti-α-Syn antibody (1:500, BD Biosciences), and a rabbit anti-α-Syn primary antibody (phospho-S129, 1:1000, Abcam) or a rabbit anti-β-actin antibody (1:10000; Abcam, Cambridge, UK) overnight at 4 °C. After incubation with the corresponding secondary antibodies for 1 h, the membranes were scanned using an Odyssey Sa imaging system (LI-COR Biosciences, Lincoln, NE, USA). The density of the results was quantified by two experimenters using ImageJ software (NIH, Bethesda, MD, USA), and the two experimenters were blinded to the characteristics of the samples studied.
Odyssey sa imaging system
Manufactured by LI COR
Sourced in United States, United Kingdom
The Odyssey Sa Imaging System is a digital imaging device designed for the detection and quantification of fluorescent signals in a variety of life science applications. It utilizes near-infrared fluorescence technology to provide high-sensitivity detection and accurate analysis of proteins, nucleic acids, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Immobilion fl membrane, by Merck Group (4 mentions)
Odyssey sa application software, by LI COR (4 mentions)
Image studio, by LI COR (4 mentions)
Irdye 800cw secondary antibody, by LI COR (4 mentions)
Odyssey blocking buffer, by LI COR (4 mentions)
Irdye 800 or 680 secondary antibodies, by LI COR (3 mentions)
Nitrocellulose membrane, by LI COR (3 mentions)
Celltag 700 stain, by LI COR (3 mentions)
Image studio software version 5, by LI COR (2 mentions)
Anti rabbitirdye 800cw secondary antibody, by LI COR (2 mentions)
Ab139690, by Abcam (2 mentions)
Ab50818, by Abcam (2 mentions)
Ab128874, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Image studio lite software, by LI COR (2 mentions)
Mts assay, by Promega (2 mentions)
Image studio software, by LI COR (2 mentions)
Col1a, by Abcam (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Rabbit anti β actin antibody, by Abcam (1 mentions)
44 protocols using odyssey sa imaging system
1
Western Blot Analysis of α-Synuclein
2
Quantitative Infrared Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All cell lysates and immunoprecipitations were analysed by LI-COR quantitative infrared western technology. Proteins were separated by SDS–PAGE and blotted onto Immobilion FL membrane (Millipore). Membranes were incubated with indicated primary antibody and subsequently with IRDye 800 or 680 secondary antibodies (LI-COR). Membranes were scanned using the Odyssey Sa imaging system (LI-COR) and quantification was carried out using the Odyssey Sa Application software (LI-COR). Uncropped scans of key western blots are found in Supplementary Fig. 3 .
3
Curcumin and Cisplatin Cytotoxicity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IC50 values for the single treatment with either curcumin and cisplatin of NSCLC cell lines were assessed by the MTS [3-(4,5-dimethylthiazol-2-yl)-2H-tetrazolium, inner salt] assay purchased from Promega (Madison, WI, USA). Tumour cells were plated at a density of 1×104 cells/well in 96-well plates and incubated overnight in humidified air with 5% CO2 at 37°C. NSCLC cells were then treated with a working concentration of curcumin (10, 20, 30 and 40 µM) and cisplatin (5, 10, 15, 20 and 25 µM) for 48 h. After a 48-h incubation, 15 µl of MTS solution was added to each well and incubated for another 4 h. Solubilisation solution (100 µl) was later added to the cells, and the absorbance at 570 nm was measured using Odyssey® SA Imaging System (Li-Cor, Lincoln, NE, USA), using wells without cells as the blank. Cell viability was calculated according to the following formula: Cell viability (%) = cells (sample)/cells (control) × 100 and IC50 was calculated using log formula.
4
SOCS3 Binding Assay using Peptide Arrays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Arrays were produced by automatic SPOT synthesis and synthesised on continuous cellulose membrane supports on Whatman 50 cellulose membranes using Fmoc-chemistry with the AutoSpot-Robot ASS 222 (Intavis Bioanalytical Instruments AG) as we have previously described76 (link). Following blocking of non-specific protein binding sites by incubation in tris-buffered saline with Tween-20 (TBST; 50 mM Tris pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween 20) containing 5% (w/v) BSA, membranes were overlaid with 10 μg ml−1 purified recombinant Trx-polyHis-tagged SOCS3 (Sino Biological Inc.) diluted in TBST-5% (w/v) BSA. After washing in TBST, bound SOCS3 was detected by probing overlays with anti-SOCS3 antibody followed by IRDye-conjugated secondary antibody prior to visualisation using a LI-COR Odyssey Sa imaging system. As a negative control, identical arrays were identically treated in parallel minus recombinant SOCS3.
5
Quantitative Western Blot Analysis
6
Western Blot Analysis of Bromodomain Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blots were probed with antibodies for Brd4 (AbCam ab128874, 1:1,000 dilution), Brd3
(AbCam ab50818, 1:500 dilution), Brd2 (AbCam ab139690, 1:2,000 dilution),
β-actin (AbCam ab8227, 1:2,000 dilution) and cMyc (AbCam ab32072, 1:1,000
dilution) antibodies. Blots were developed with anti-Mouse or anti-Rabbit
IRDye® 800CW secondary antibody from Licor (1:10,000 dilution) and bands
visualized using Licor Odyssey Sa imaging system. Image processing and band
intensity quantification were done using Licor Image Studio software Version
5.2.5. Ubiquitination blots were probed with anti-6×His antibody (AbCam
ab18184, 1:2,000 dilution) and then with anti-Mouse IgG, HRP-linked antibody
(Cell Signaling Technology #7076, 1:2,000 dilution). Probed blots were
visualised with ECL Western Blotting Substrate (Pierce #32106) on film.
(AbCam ab50818, 1:500 dilution), Brd2 (AbCam ab139690, 1:2,000 dilution),
β-actin (AbCam ab8227, 1:2,000 dilution) and cMyc (AbCam ab32072, 1:1,000
dilution) antibodies. Blots were developed with anti-Mouse or anti-Rabbit
IRDye® 800CW secondary antibody from Licor (1:10,000 dilution) and bands
visualized using Licor Odyssey Sa imaging system. Image processing and band
intensity quantification were done using Licor Image Studio software Version
5.2.5. Ubiquitination blots were probed with anti-6×His antibody (AbCam
ab18184, 1:2,000 dilution) and then with anti-Mouse IgG, HRP-linked antibody
(Cell Signaling Technology #7076, 1:2,000 dilution). Probed blots were
visualised with ECL Western Blotting Substrate (Pierce #32106) on film.
7
Western Blot Analysis of Bromodomain Proteins
8
Protein expression analysis in cell extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested and homogenized in RIPA buffer (Sigma-Aldrich® GmbH, St. Louis, MO, USA) containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40, 0.1% (v/v) sodium dodecyl sulfate and protease, phosphatase inhibitor (Roche, Basel, Switzerland). Protein concentrations were determined by DC™ Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and equal amounts of proteins (25 µg) were separated by 8% and 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with antibodies specific to Myosin (1:200), MuRF-1 (1:200), and Foxo (1:200) (all from Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (1:10,000, Acris Antibodies GmbH, Herford, Germany), respectively, and appropriate secondary dye-conjugated antibodies goat anti-mouse IRdye800, IRdye650 (1:10,000; LI-COR®, Lincoln, NE, USA) to reveal protein bands for 1 h at room temperature. Membranes were scanned using the Odyssey SA Imaging System (LI-COR®, Lincoln, NE, USA). Experiments were performed in duplicates and the signal intensity was normalized to GAPDH.
9
Western Blot Protein Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein concentration was measured with BCA Protein Assay Kit (C503021, Sangon Biotech, Shanghai, China). 20 μg protein per sample was resolved on polyacrylamide gels and transferred to 0.22 μm nitrocellulose filter membranes. Membranes were blocked with 5% nonfat milk (E-BC-R337, Elabscience Biotechnology Co., Ltd.) in Tris-buffered saline (TBS) for 1 h at room temperature, and then with primary antibody in TBS supplemented with 3% BSA overnight at 4 °C. Membranes were washed with TBST (3 × 10 min), then incubated with the IRDye® 800CW Goat anti-Rabbit IgG Secondary Antibody or IRDye® 680RD Goat anti-Mouse IgG Secondary Antibody (926-32211 and 926-68070, LI-COR Biosciences, Lincoln, NE, USA) in TBS at room temperature for 2 h and washed again. Protein imaging was performed on an odyssey Sa Imaging System (LI-COR Biosciences) and quantification was conducted on an Image Studio Lite software (Ver 5.2, LI-COR Biosciences).
10
Quantifying Estrogen Receptor Alpha in MCF7:WS8 Cells
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!