Quantification of protein expression from cell lysates was performed by Western blot, as previously described (46 (link)). For that, primary rabbit anti-human antibodies from Bio-Rad, Stockholm, Sweden (ACE2 #AHP888) and Cell Signaling, Danvers, Massachusets, USA (GAPDH #5174S) were used at dilution 1:1000, and an anti-rabbit secondary antibody #09/2029 lot. 28 (Cell Signaling, Danvers, Massachusets, USA) conjugated to HRP was used. Optical density was detected using a LI-COR odyssey Fc imager system (LI-COR, Lincoln, USA). Optical density ratio between samples and GAPDH were calculated and normalized towards poly(I:C) stimulated samples.
Odyssey fc imager system
Manufactured by LI COR
Sourced in United States
The Odyssey Fc imager system is a high-performance fluorescence and chemiluminescence imaging platform designed for a wide range of imaging applications. It features advanced optics, sensitive detectors, and flexible software to capture high-quality images of gels, blots, and other samples.
Automatically generated - may contain errors
Lab products found in correlation
Image studio, by LI COR (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
C myc, by Abcam (1 mentions)
Hrp conjugated anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Anti tubulin, by Cell Signaling Technology (1 mentions)
Anti rad9, by Santa Cruz Biotechnology (1 mentions)
Anti rad1, by Santa Cruz Biotechnology (1 mentions)
Anti topbp1, by Santa Cruz Biotechnology (1 mentions)
Anti chk2 pt68, by Cell Signaling Technology (1 mentions)
Anti rad17, by Santa Cruz Biotechnology (1 mentions)
Anti chk1 ps345, by Cell Signaling Technology (1 mentions)
Anti γh2ax, by Merck Group (1 mentions)
Anti chk1, by Cell Signaling Technology (1 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Ab129153, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
7 protocols using odyssey fc imager system
1
Quantification of Protein Expression via Western Blot
2
Quantification of PRR Proteins in Lung
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed for the quantification of PRR expression in lung. After total protein determination in lung homogenates, equal amount of each sample (20 μg protein) was loaded and electrophoresed on a 10 % polyacrylamide gel. The fractionated protein was blotted onto a PVDF membrane and blocked in blocking buffer (5 % non-fat dry milk, TBS-T). The membrane was then incubated in primary antibody; anti-RIG-I, anti-MDA5 or anti-TLR3. As loading control, an antibody was used directed towards the housekeeping protein β-tubulin. Appropriate secondary antibody was used directed towards each primary antibody, shown in Additional file 2 : Table S2 as supplementary data. Proteins of interest were detected with detection kit SuperSignal®West Femto Maximum Sensitivity Substrate (Fischer Scientific AB, Sweden) and after exposure to chemiluminescence using LI-COR Odyssey Fc Imager system (LI-COR, Biosciences) and the software ImageStudio (2012 LI-COR Inc© version 3.1.4). The band density was calculated from the optical density and ratio was obtained after calculations in Microsoft Excel.
3
Butyrate Modulates Protein Expression in Colon Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates for Western blotting were prepared as previously described [17 (link)] with minor modifications. Briefly, HCT116, HT-29, and Caco-2 cells in 60 mm culture dishes were treated with butyrate (0 to 4 mM) when cells reached ~30–40% confluency. Cells were harvested and lysed 24 h after butyrate treatment using cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA). Bradford assay was performed to determine the protein concentration. Protein samples were separated with SDS-page gel electrophoresis using gradient gels (4 to 20%) and transferred onto PVDF membranes. After blocking the membranes with 5% dry milk, membranes were incubated overnight at 4 °C with the following primary antibodies: phospho-p44-42 (ERK1/2) T-202/Y-204, ERK1/2, p21, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Inc., Danvers, MA, USA), and c-Myc (Abcam, Cambridge, MA, USA). After TBS wash, membranes were incubated for 1 h with anti-rabbit HRP-conjugated secondary antibody (1:5000 dilution) (Cell Signaling Technology, Inc., Danvers, MA, USA). Finally, these membranes were TBS-washed and then incubated with chemiluminescence (ECL) reagent; and protein images were visualized and quantified using a LI-COR Odyssey Fc imager system (Lincoln, NE, USA).
4
Western Blot Analysis of DNA Damage Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analyses were performed with anti-γ-H2AX (Millipore), anti-H2AX (Sigma-Aldrich), anti-RAD9 (Santa Cruz or Bethyl), anti-RAD17, anti-RAD1, anti-TopBP1, anti-CLASPIN (Santa Cruz), anti-tubulin, anti-CHK1, anti-CHK1-pS345, and anti-CHK2-pT68 (Cell Signaling Technology) antibodies. The antibodies against the phospho-peptides (pThr292: LQAHSpTPHPDC, pThr313: CAMETpTIGNEG, pSer326: CEGSRVLPSIpS) were raised by MBL (Medical and Biological Laboratories (MBL), Japan). HRP-conjugated antibodies were used as secondary antibodies, the ECL signal was incorporated using a Licor Odyssey Fc imager system, and the quantification was performed with the Image Studio software.
5
HMGA1 Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cytoplasmic and nuclear protein extracts were prepared according to our previous study.6 (link) Briefly, proteins were extracted using the NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific), separated by SDS-PAGE gel, and transferred to immune-blot polyvinylidene fluoride (PVDF) membrane. The membrane was then probed with 1:30,000 dilution of rabbit anti-HMGA1 antibody (Abcam #ab129153) as primary antibody and 1:1,000 dilution of goat anti-rabbit IgG as secondary antibody (Abcam #ab205718). An enhanced chemiluminescence detection system (ECL, GE Health Care) was used to detect target proteins, and the image was captured by a LI-COR Odyssey Fc Imager system. To ensure equal loading of the proteins in each well, membranes were re-probed with anti-TATA binding protein antibody (Abcam #ab197874).
6
Protein Expression Analysis of Pancreatic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from pancreatic tissue and HPDE6C7 cells using a RIPA buffer with 1% phenylmethylsulfonyl fluoride. Total protein concentration was quantified using the Bicinchoninic Acid Protein Assay kit (Wuhan Boster Biological Technology Co., Ltd.). Protein samples (30 µg/well) were transferred onto polyvinylidene fluoride membranes following 10% and 15% SDS-PAGE. Membranes were blocked with 5% skimmed milk for 45 min at room temperature. The membranes were subsequently incubated with primary antibodies, including those for TRAF6 (1:2,000; cat. no. ab33915; Abcam), NLRP3 (1:1,000; cat. no. IMG-6668A; Novus Biologicals, Inc.), caspase-1 (1:1,000; cat. no. ab179515; Abcam), caspase-3 (1:1,000; cat. no. ab14220; Cell Signaling Technology, Inc.) and β-actin (1:5,000; cat. no. ab6276; Abcam) overnight at 4°C. After washing with TBS-0.1% Tween-20 buffer, membranes were incubated with goat anti-rabbit IgG (1:10,000; cat. no. ab4413; Cell Signaling Technology, Inc.) at room temperature for 1 h. Blots were scanned using the Odyssey® Fc Imager system (LI-COR Biosciences), and protein expression was semi-quantified using imageJ software (version 1.4.1; National Institutes of Health) with β-actin as the loading control.
7
Immunoblotting of BRD4 and β-actin
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!