Cytosolic and mitochondrial proteins of interest were analyzed by Western blot method as described previously (Sadigh-Eteghad et al., 2015 (link)). Briefly, proteins were separated using 12.5% polyacrylamide gel and transferred onto a poly-vinylidene-difluoride (PVDF) membrane (Roche, United Kingdom). Membranes were incubated with anti-SIRT1 (sc-15404), PGC1-α (sc-5815), NRF1 (sc-101102), TFAM (sc-166965), iNOS (sc-8310), TNF-α (ab6671), and IL-1β (sc-7884) primary antibodies in 1:500 concentration. Finally, membranes were placed in ECL prime Western blotting detection reagent (Amersham, United Kingdom). Signals were visualized by exposure to autoradiography film (Kodak, United States). Anti-GAPDH (sc-32233) and cytochrome c (sc-13156) antibodies were used for internal control of cytosolic and mitochondrial proteins, respectively. The signal intensity of each band was quantitated using ImageJ 1.62 software (National Institutes of Health, United States).
Autoradiography film
Manufactured by Kodak
Sourced in United States
Autoradiography film is a specialized photographic film used in the laboratory for the detection and visualization of radioactive signals. It is designed to capture and record the distribution and intensity of radioactive isotopes that have been incorporated into biological or chemical samples. The film provides a precise, high-resolution image of the labeled components, enabling researchers to analyze the data and draw conclusions about the underlying biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
1 14c acetate, by Cytiva (2 mentions)
High performance tlc silica gel 60 plates, by Merck Group (2 mentions)
Phosphorimager screen, by Cytiva (2 mentions)
Imagequant software, by Cytiva (2 mentions)
Image pro plus software 6, by Media Cybernetics (2 mentions)
Ecl detection reagent, by GE Healthcare (2 mentions)
Cdp star, by Merck Group (2 mentions)
Un scan it gel software, by Silk Scientific (2 mentions)
Pvdf membrane, by Roche (1 mentions)
Ecl prime western blotting detection reagent, by Cytiva (1 mentions)
Chemiluminescent detection system, by Beyotime (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Gapdh, by Abcam (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Beyotime (1 mentions)
β actin mouse monoclonal antibody, by Beyotime (1 mentions)
Bovine cam, by Merck Group (1 mentions)
18 protocols using autoradiography film
1
Western Blot Analysis of Cytosolic and Mitochondrial Proteins
2
Krox20 Protein Binding Analysis by EMSA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For electrophoretic mobility shift assays (EMSA), the Krox20 protein was produced with the TNT (T7)-coupled in vitro transcription/translation system (Promega, Madison, WI, USA) as previously described [12 (link)]. The Probes used for KROX binding corresponded to the -513 and -136 KROX binding sites (Table 1 ). For EMSA, 3 or 9-μL of in vitro translated Krox20 protein were mixed in 20-μL binding reaction containing 20% glycerol, 50 mM Tris-HCl pH 7.5, 250 M NaCl, 2.5 mM EDTA, 2.5 mM DTT, and 5 mM MgCl2. The reactions were incubated on ice for 10 min before the addition of 10,000 counts/min [α-32P]dATP-labeled oligonucleotides. Mixtures were further incubated on ice for 30 min before being loaded on a 0.5× Tris-boric acid-EDTA buffer-4% polyacrylamide gel, and then electrophoresis was carried out at 250 V for 1.5 h at 4 °C. The gel was dried and exposed to a Kodak autoradiography film overnight at −80 °C.
3
Bacterial Lipid Composition Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The lipid compositions of bacterial strains were determined following labeling with [1-14C]acetate (Amersham Biosciences). Cultures (1 ml) of Burkholderia s.l. strains were inoculated from precultures grown in the same medium. After addition of 1 μCi of [14C]acetate (60 mCi mmol-1) to each culture, the cultures were incubated overnight. Cells were harvested by centrifugation, washed with 500 μl water once, resuspended in 100 μl water, and then lipids were extracted according to Bligh and Dyer (Bligh and Dyer, 1959 (link)). Aliquots of the lipid extracts were spotted on high performance TLC silica gel 60 plates (Merck, Poole, UK) and separated in two dimensions using chloroform/methanol/water (16:4:1, v/v/v) as a mobile phase for the first dimension and chloroform/methanol/acetic acid (15:3:2, v/v/v) as a mobile phase for the second dimension (Tahara and Fujiyoshi, 1994 (link)). To visualize membrane lipids, developed two-dimensional TLC plates were exposed to autoradiography film (Kodak) or to a PhosphorImager screen (Amersham Biosciences). The individual lipids were quantified using ImageQuant software (Amersham Biosciences) (Vences-Guzmán et al., 2011 (link)).
4
Western Blot Analysis of MTDH Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from cells using RIPA lysis buffer with proteinase inhibitor. Concentrations of total cellular protein were determined using a BCA assay kit (Beyotime, Jiangsu, China). Then, 20 μg of protein mixed with 2× SDS loading buffer was loaded per lane, separated by 10% SDS-PAGE, and electrophoretically transferred onto a nitrocellulose membrane (Bio-Rad, Munich, Germany). Antibodies to MTDH (1:1000; Abcam, Cambridge, United Kingdom) and GAPDH (1:2500; Abcam) were incubated with the membranes overnight at 4°C. The membranes were washed and incubated with horseradish peroxidase–conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology, California). The results were visualized with a chemiluminescent detection system (Beyotime) and exposed with an autoradiography film (Kodak, Shanghai, China). Protein levels were quantified by density analysis using Quantity One software (Bio-Rad).
5
Western Blot Analysis of Protein Extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cell extracts were dissolved in SDS-loading buffer, and the lysate (20 μg of protein) was electrophoresed on a 12% SDS polyacrylamide gel (SDS-PAGE). The separated proteins were transferred for 90 min at 90 V to nitrocellulose transfer membranes (BD Biosciences, San José, CA) and then blocked for 1 h with 5% skimmed milk. Primary antibodies (Table 1 ) were incubated overnight at 4°C. After several washes, horseradish peroxidase-conjugated goat anti-mouse or swine anti-rabbit immunoglobulin (DakoCytomation, Denmark) was added for 90 min at room temperature. Protein bands were revealed using an enhanced chemiluminescence substrate (Biological Industries, Reactiva, Barcelona, Spain) and recorded on autoradiography film (Kodak Rochester, NY, USA). WB analysis was performed by digital scanning of the blots, followed by densitometric analysis with ImageJ [36 ]. Analysis of p63 was normalized to tubulin as the loading control.
6
Quantitative Western Blot Analysis of ATF3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HepG2 cells were lysed in ice-cold lysis buffer [150 mM NaCl, 100 mM Tris (pH 8.0), 1% Tween-20, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml trypsin, and 10 µg/ml leupeptin]. The lysate was left on ice for 20 min and then centrifuged at 4°C, 1,200 × g for 10 min. The clarified supernatant was collected, and the protein concentration was measured by using a Biotek protein assay kit (Elx800; BioTek Instruments, Winooski, VT, USA). Protein lysate (60 µg per well), was separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were incubated with 5% non-fat milk for 1 h at room temperature and then with mouse monoclonal anti-human ATF3 antibody (1:100 dilution, ab58668; Abcam, Cambridge, MA, USA) at 4°C overnight. The membranes were washed and stained with a horseradish-peroxidase-conjugated secondary antibody (at 1:1,000 dilution, A0216, Beyotime Institute of Biotechnology, Shanghai, China). Proteins were visualized using Enhanced Chemiluminescence Plus system (Beyotime Institute of Biotechnology) and exposed to autoradiography film (Kodak, Rochester, NY, USA). Blots with mouse monoclonal β-Actin antibody (at 1:1,000 dilution, AA128; Beyotime Institute of Biotechnology) were similarly generated to ensure that equal amounts of protein were loaded in the wells.
7
CCaMK Autophosphorylation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The autophosphorylation assay was performed in 10 μl reaction mixture using 0.4 μg of CCaMK protein and its mutated versions. The reaction buffer contained 50 mM HEPES pH 7.5, 10 mM magnesium acetate, 1 mM DTT, 10 μM ATP and 0.5 μCi/μl [γ-32P] ATP, in the presence of 5 mM EGTA with or without 1 μM of bovine brain CaM (Sigma); and 0.5 mM of CaCl2 with or without bovine CaM. Samples were incubated at 30°C for 30 min. To stop the reaction, SDS–PAGE sample buffer was added, followed by boiling the samples for 2 min. Samples were separated by a 12.5% SDS–PAGE. Protein gel was then dried and exposed to autoradiography film (Kodak).
8
Western Blot Visualization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were transferred to a nitrocellulose membrane using an iBlot system as per the manufacturer's instructions (Life Technologies). Membranes were then blocked in PBST with non‐fat dried milk (5%) for 1 h prior to probing with anti‐mCherry, anti‐GFP or anti‐β‐tubulin primary antibodies overnight. Post incubation, membranes were washed 3x in PBST. Bound antibodies were detected using HRP‐secondary antibody (Dako) in PBST with milk (1 h). Membranes were then washed 3× in PBST, rinsed in deionized water and finally subjected to enhanced chemi‐luminescence by incubation in freshly‐prepared visualization solution (2 min). The membrane was exposed to autoradiography film (Kodak) for 3 to 45 seconds, as appropriate.
9
Quantification of CUG Repeat RNA
10
Western Blot Analysis of Hippocampal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen (−80°C) hippocampal tissues were homogenized and equal amounts of proteins were resolved on polyacrylamide gel, then transferred to PVDF membranes (Millipore). Membranes were blocked and then probed with primary antibodies against NR2B (H-50, sc-9057, from Santa Cruz Biotechnology), BACE1 (D10E5), CREB, pCREB, CaMKII, pCaMKII, GAPDH (all from Cell Signaling Technology), sAPPβ (IBL) or β-CTF (6E10, Signet, see Table 3 ) overnight at 4°C. Membranes were then incubated with an HRP-conjugated secondary antibody (ZSGB-BIO or CST, see Table 3 ) at room temperature. Protein bands were detected by ECL detection reagent (RPN2232; GE Healthcare) and captured on an autoradiography film (Kodak). Integrated optical density was determined using Image-Pro Plus software 6.0 (Media Cybernetics). Standard curves were constructed to establish that we operated within the linear range of the detection method. Co-detection of GAPDH on the same membrane served as a loading control. Quantitative analysis was performed by experimenters blind to the treated and/or genotype group.
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!