Final concentration 2 nM of radiolabelled RNA was adjusted to 5 ml with Milli-Q water, incubated 1 min at 95 °C and cooled on ice for 2 min. RNA was allowed to fold for 30 min at 37 °C in binding buffer (mM Tris-HCl pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.1 mM CaCl2, 1 mM DTT, 0.1 mg/ml BSA, 0.4 mg/ml fragmented yeast RNA (Sigma), 5% glycerol, 0.025% bromophenol blue and 0.025% xylene cyanol). Recombinant YBX1 (Abcam) was added at the concentrations indicated and the binding reaction was carried out at 30 °C for 30 min. Samples were loaded on a non-denaturing 0.7% agarose gel in cold 1× TBE buffer. After 2 h at 120 V of gel electrophoresis, the gels were vacuum dried for 90 min at 80 °C and exposed to an Amersham Hyperfilm ECL film.
Hyperfilm ecl film
Manufactured by Cytiva
Sourced in United States
Hyperfilm ECL is a high-performance X-ray film designed for chemiluminescent detection. It is optimized for use with enhanced chemiluminescence (ECL) detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant ybx1, by Abcam (2 mentions)
Eluate only, by Thermo Fisher Scientific (2 mentions)
Oti4c6, by OriGene (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal antibody directed against β actin, by Merck Group (1 mentions)
Tris glycine running buffer, by Bio-Rad (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Ponceau s staining, by Merck Group (1 mentions)
Perfecthyb plus, by Merck Group (1 mentions)
Zeta probe membrane, by Bio-Rad (1 mentions)
Protein standard, by Bio-Rad (1 mentions)
Sds page reagents, by Bio-Rad (1 mentions)
Hsp70, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Sds sample buffer, by Bio-Rad (1 mentions)
Enhanced chemiluminescence ecl reagent, by Cytiva (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Hsp90, by Cell Signaling Technology (1 mentions)
15 protocols using hyperfilm ecl film
1
RNA Folding and YBX1 Binding Assay
2
Quantifying Hypothalamic Glucocorticoid Receptor
3
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were extracted from cell culture using RIPA buffer. They were then sonicated for a duration of 10–30 sec, followed by a 5-min incubation at 98°C in 1× Laemmli buffer (Alfa Aesar). Subsequently, these treated samples were loaded onto 4%–15% Mini-PROTEAN TGX gels (Bio-Rad) and electrophoresed in Tris-glycine running buffer (Bio-Rad) under a constant voltage of 100 V for a period of 1 h. For the purpose of transferring proteins onto nitrocellulose membranes, gels were transferred over a span of 1 h 30 min at a consistent voltage of 15 V. The successful transference of proteins to the membranes was assessed using Ponceau S staining (Merck). The nitrocellulose membranes were then subjected to blocking, which was performed for 1 h at room temperature using 5% milk in PBS with 0.1% Triton X-100 (PBST). Following blocking, the membranes were incubated overnight at 4°C with primary antibodies. After primary antibody incubation, the membranes underwent three PBST washes and were subsequently incubated with secondary antibodies for 1 h at room temperature. After an additional three PBST washes, protein bands were visualized using an enhanced chemiluminescence (ECL) detection, and the results were recorded on Amersham Hyperfilm ECL film.
4
Rolling Circle Amplification of Genomic DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted from cells (106 cells per sample) using the QIAGEN Core B kit and re-suspended in 20 mM Tris-HCl. 30 ng of genomic DNA was amplified in a PCR reaction containing φ29 polymerase, 1% Tween-20, 200 μg/mL BSA and dTTP, dGTP and dATP for 8 h at 30°C followed by 20 min at 65°C. PCR reactions were run with and without φ29 polymerase to ensure the signal was specific for rolling-circle amplification products. Amplified samples were then blotted onto Zeta-Probe membrane (Bio-Rad) using a slot blotter. DNA was crosslinked to the membrane with a UVA Stratalinker 2,400 and membranes were then soaked in PerfectHyb Plus (Sigma Aldrich) for 20 min at room temperature. A 3’ Digitonin (DIG) tagged [CCCATT]5 oligonucleotide was then diluted in PerfectHyb to a final concentration of 40 nM in 20 mL hybridization buffer, and was hybridized with the membrane for 2 h at 37°C. Following hybridization, membranes were briefly washed twice with wash buffer (0.1 M Maleic acid, 3 M NaCl; 0.1% Tween 20 adjusted to pH7.5), before blocking for 30 min and probing with a DIG antibody for 30 min. Finally, membranes were washed 3 times with wash buffer before placing into a cassette with CDP-Star solution. Blots were then developed onto Amersham Hyperfilm ECL film.
5
Endoplasmic Reticulum Stress Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibody anti-β-actin, BSA, FCS, HBSS, l-glutamine, penicillin-streptomycin, PBS, propidium iodide (PI), Q, RNAse, RPMI-1640, and tunicamycin were from Sigma-Aldrich (St. Louis, MO, USA). Antibodies anti-BiP, -caspase 3, -CHOP, -Hsp70, -Hsp90, -IRE1α, and -PARP were from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase- (HRP-) conjugated anti-rabbit- and anti-mouse-immunoglobulin antibodies, enhanced chemiluminescence (ECL) reagents, and Hyperfilm-ECL film were from Amersham (Arlington Heights, IL, USA). Lipofectamine RNAiMAX and OPTI-MEM medium, small interfering- (siRNA-) IRE1, and relative scrambled siRNA (Ambion) were from Life Technologies (Invitrogen, San Giuliano Milanese, Italy). 2-Phenylethynesulfonamide (PES) and 4μ8c were from Merck (INALCO, Milan, Italy). RC-DC protein assay, SDS-sample buffer, protein standard, SDS-PAGE reagents, and polyvinylidene difluoride (PVDF) membranes were from Bio-Rad Laboratories (Segrate, Italy). Antibody anti-tubulin, siRNA-Hsp70, and scrambled siRNA were purchased from Santa Cruz Biotechnology (Tebu-Bio, Magenta, Italy). Thapsigargin and z-VAD.fmk were from Calbiochem (San Diego, CA, USA). Other reagents were of the highest purity and purchased from Bio-Rad, Invitrogen, or Sigma.
6
In Vitro Kinase Assay of YBX1 and RSK1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One microgram of recombinant YBX1 (Abcam) was incubated with 20 ng of recombinant RSK1 protein (R&D) and the corresponding in vitro-transcribed RNA in kinase buffer (25 mM TRIS/HCl, pH 7.5, 5 mM glycerolphosphate, 2 mM DTT, 10 mM MgCl2, 50 µM ATP, 10 µCi γ-32P (Perkin Elmer), protease inhibitor mixture Complete (Roche), phosSTOP (Roche)) for 15 min at 30 °C. Laemmli buffer was added to stop the reaction and boiled at 95 °C for 5 min. Proteins were separated by electrophoresis in 4–12% NuPAGE Bis–Tris gels (Invitrogen). Subsequently, the gel was dried at 80 °C for 1 h and then exposed to an Amersham Hyperfilm ECL film. The band intensities were measured using ImageJ/Fiji software v2.0.0.
7
Western Blot Analysis of Histone Modifications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA buffer (50 mM Tris, 150 mM NaCl, 0.5% Deoxycholate, 1% NP-40, 0.1% SDS, 1 mM DTT, and 1% protease/phosphatase inhibitor) and supernatant was cleared by centrifugation. Protein concentration was determined using the Pierce BCA assay kit (Thermo, 23227). Between 40mg and 120mg of each protein sample was boiled in Laemmli buffer with 10% β-Mercaptoethanol and equal percentages of each sample were run on 4-15% polyacrylamide gels (BioRad, 4561086). Resolved proteins were wet-transferred to nitrocellulose membranes (Amersham), which were then blocked in a 5% BSA (VWR, 9048-46-8) solution made in 1x TBST buffer (20 mM Tris base, 0.15 M NaCl, 0.1% Tween, adjusted to pH 7.6). Membranes were then incubated with antibody solutions prepared in 5% BSA overnight. Antibodies used were (H3K27me3 Cell Signaling C26B11 1:500, EZH2 Cell Signaling 5246S 1:200, B2M Millipore MABF1968 1:2000, HLA-DR,DQ,DP AbCAM ab7856 1:500, Total Histone H3 AbCAM ab1791 1:5000). After washing, secondary antibodies were added (Novus anti-Rabbit-HRP and anti-Mouse-HRP), incubated, and washed. Bands were visualized with West Plus Pico ECL (Thermo) and exposed to Hyperfilm™ ECL™ film (Amersham). Protein molecular weights were determined with Precision Plus Protein™ Kaleidoscope™ Prestained Protein Standards (BioRad).
8
SDS-PAGE and Immunoblotting of Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were separated by 4–20% SDS-PAGE and either silver stained (eluate only; ThermoFisher Scientific) according to manufacturer’s protocols, or transferred to PVDF membranes at 0.25 A for 1.5 hrs at 0°C. Transferred PVDF membranes were blocked in 5% milk powder in 0.2% Tween-TBS overnight at 4°C and immunoblotted with mouse anti-FLAG-HRP (Sigma) conjugated primary antibody, rabbit α-ELAC2 (RNZ2) polyclonal antibody (Proteintech, 10071–1-AP, 1:2,000) and mouse α-PUS7 monoclonal antibody (OriGene, OTI4C6; 1:1,000) followed by goat α-rabbit, and α-mouse secondary antibodies (BioRad; 1:10,000), respectively. Pierce ECL Western Blotting Substrate (Thermo Scientific) was used to detect bands with Hyperfilm ECL film (Amersham).
9
SDS-PAGE Protein Separation and Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were separated by 4 to 20% SDS-PAGE and either silver stained (eluate only; ThermoFisher Scientific) according to manufacturer’s protocols or transferred to PVDF membranes at 0.25 A for 1.5 h at 0 °C. Transferred PVDF membranes were blocked in 5% milk powder in 0.2% Tween-TBS overnight at 4 °C and immunoblotted with mouse anti-FLAG-HRP (Sigma) conjugated primary antibody, rabbit anti-ELAC2 (RNZ2) polyclonal antibody (Proteintech, 10071-1-AP, 1:2000) and mouse anti-PUS7 monoclonal antibody (OriGene, OTI4C6; 1:1000) followed by goat anti-rabbit, and anti-mouse secondary antibodies (BioRad; 1:10,000), respectively. Pierce ECL Western Blotting Substrate (Thermo Scientific) was used to detect bands with Hyperfilm ECL film (Amersham).
10
Western Blot Analysis of Protein Degradation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were separated by SDS-PAGE on NuPage 4–12% Bis-Tris gels and transferred to Amersham Protran 0.45 NC nitrocellulose membranes (GE Healthcare) using wet transfer. Membranes were blocked using 5% w/v milk in Tris-buffered saline (TBS) with 0.1% Tween-20. Blots were probed using anti-VHL (CST-68547), anti-CRBN (Novus, NBP1-91810) and anti-β-tubulin hFAB-rhodamine (BioRad, 12004166) primary antibodies, followed by incubation with secondary anti-Rabbit IRDye 800CW (ab216773) or anti-rabbit HRP-conjugated (CST-7074) antibodies. Blots were developed using a Bio-Rad ChemiDoc MP Imaging System or the Amersham ECL Prime western blotting detection kit and Amersham Hyperfilm ECL film, as appropriate. Band quantification was performed using the ImageJ software. Band intensities were normalized to the β-tubulin loading control and reported as % of the average 0.1% DMSO vehicle intensity. Degradation data was plotted and analysed using Prism (Graphpad, version 7). DC50 values (concentration to reach 50% maximal degradation) were estimated by fitting band intensity against log[concentration]. Apparent half-life values (time to reach 50% maximal degradation) were estimated by fitting band intensity against time using a single-phase exponential decay model.
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!