The SDS-PAGE was conducted using the Mini-PROTEAN® System (Bio-Rad, Hercules, CA, USA). The sunflower seed meal protein samples (2 mg) were dissolved in 0.5 mL of sample buffer (0.08 M Tris-HCl buffer, pH 6.8), 1% (w/v) SDS, 2% (v/v) 2-β-mercaptoethanol, 5% (v/v) glycerol, and 0.025% (w/v) bromophenol blue and mixed well [27 (link)]. The protein was subjected to SDS-PAGE analysis using a 5% concentrating gel and a 12% separating gel. A marker with a molecular weight range of 11~180 kDa (Sigma-Aldrich Co., St. Louis, MO, USA) was employed. 4 μL samples of supernatant were applied to the stacking gel slot and 1 L of electrode buffer was added. Electrophoresis was conducted initially at 30 V for 1.5 h, followed by a change to 60 V for an additional 2 h. After electrophoresis, the gel was stained with Coomassie brilliant blue R250. The decolorization was achieved using a methanol-ice acetic acid solution, and the photographic analysis was performed after decolorization. The images were finally taken using a Bio-Rad gel imaging system. Image Studio Lite (version 5.2, LI-COR Biosciences, Lincoln, NE, USA) The intensity of the stained bands is analyzed by Image Studio Lite to determine the relative ratio of specific proteins to the total protein content, providing an indication of the component purity of the sample.
Image studio lite
Manufactured by LI COR
Sourced in United States, United Kingdom
Image Studio Lite is a software application that enables the analysis and quantification of fluorescent and chemiluminescent Western blot, gene expression, and other types of gel-based images. It provides basic image processing and analysis tools to assist researchers in their scientific investigations.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (35 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (26 mentions)
Odyssey clx, by LI COR (25 mentions)
Odyssey imaging system, by LI COR (23 mentions)
Odyssey clx imaging system, by LI COR (23 mentions)
Odyssey blocking buffer, by LI COR (19 mentions)
Ripa buffer, by Thermo Fisher Scientific (16 mentions)
Nitrocellulose membrane, by Bio-Rad (15 mentions)
Protease inhibitor cocktail, by Roche (14 mentions)
Odyssey fc imaging system, by LI COR (13 mentions)
Odyssey imager, by LI COR (11 mentions)
Pvdf membrane, by Merck Group (11 mentions)
Irdye 800cw, by LI COR (10 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (10 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (9 mentions)
β actin, by Merck Group (9 mentions)
Protease inhibitor, by Roche (9 mentions)
Prism 7, by GraphPad (9 mentions)
Trans blot turbo transfer system, by Bio-Rad (8 mentions)
596 protocols using image studio lite
1
Sunflower Seed Meal Protein Analysis
2
Protein Expression Analysis in Cell and Tumor Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell and tumor lysates were prepared in NP-40 lysis buffer with protease inhibitor cocktail or Cell Lysis Buffer (Cell signaling, Danvers, MA, USA) respectively. Analysis of lysates was carried out as previously described [7 (link)]. Primary antibodies used were: γH2AX (Cell Signaling), RAD51 (abcam, Cambridge, MA, USA), Ku80 (Bethyl Laboratories, Montgomery, TX, USA), BRD4 (Cell Signaling), BRD2 (Cell Signaling), vinculin (Santa Cruz Biotech, Dallas, TX, USA), GAPDH (Cell Signaling), β-actin (Cell Signaling), α-Tubulin (Cell Signaling), p21 (Cell Signaling), Cleaved PARP (Cell Signaling). Blots were quantitated using ImageStudio Lite (LI-COR Biosciences, Lincoln, NE, USA).
3
Quantification of β2-Microglobulin in C. elegans
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Worms were collected at the first or fifth day of adulthood, in M9 buffer (45 mM KH2PO4, 42 mM Na2HPO4, 85 mM NaCl, 1 mM MgSO4 in water) and lysed by sonication in lysis buffer (25 mM Tris-HCl pH 7.5, 5 mM NaCl, 5 mM EDTA, 1 mM DTT, protease inhibitor cocktail Roche Applied Science). For each lysate, equal amounts of total proteins, quantified with the Pierce BCA Protein Assay Kit (ThermoScientific), were loaded onto either a 4–20% Mini-PROTEAN TGX (Biorad) or 8–18% Excel SDS gel (GE Healthcare) for electrophoresis performed under reducing conditions. Proteins were transferred to Immobilon P membranes (Millipore) and blocked with 5% non-fat milk, in tris-buffered saline and Tween 20 (TBS-T), for one hour. Western blots were developed with 4.6 μg/ml rabbit polyclonal anti-human β2-m antibody (A0072, Dako) O.N. at 4 °C and 1.3 ng/ml anti-rabbit IgG peroxidase conjugate (A0545 Sigma) for 1 h RT, as primary and secondary antibody respectively. To normalize the content of total protein, western blot was developed with 0.185 μg/ml anti-glyceraldehyde 3-phosphate dehydrogenase antibody (anti-GAPDH selected as loading control, ab181602 Abcam) O.N. at 4 °C, and 1.3 ng/ml secondary anti-rabbit IgG peroxidase conjugate (A0545 Sigma) antibody for 1 h RT. Immunoreactive bands were detected by ECL chemio-luminescence (Millipore), and quantified with Image Studio Lite (LI-COR Biosciences).
4
Western Blot Analysis of Androgen Receptor
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblots were performed as previously described (7 (link)). Membranes were incubated with the primary antibody (androgen receptor, details in Table 4 ) overnight at 4 °C. Thereafter, they were washed 3 × in Tris buffered saline-Tween 20. Membranes were then incubated with secondary antibodies for 1 hour at room temperature and fluorescent signals measured using the Odyssey CLX, LI-COR (LI-COR Biosciences, UK) scanner and analyzed using ImageStudioLite (LI-COR Biosciences, UK) software. Alpha tubulin (Sigma Aldrich, UK; 1:10,000, 3% BSA/Tris buffered saline-Tween 20) was used on all membranes as an internal housekeeping loading control protein. Results were normalized according to cellular protein levels. Results are expressed in arbitrary units.
5
Barley Transcriptome Analysis and amiRNA Design
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic sequences for HvMADS27 and HvMIR444c were obtained from the Ensembl Plants database3 and aligned with the cDNA sequence using MAFFT version 7.4 Results from transcriptome sequencing were quality checked using the FASTQC program, and adaptor trimming was performed using Trimmomatic software. All obtained reads were mapped to the barley genome and transcriptome using HISAT2 and SALMON programs, respectively, and statistical analysis was performed in R studio environment using DESeq2 software (Love et al., 2014 (link)). Thereafter, the online tool WMD3-Web MicroRNA Designer5 was used to prepare construct overexpressing artificial miRNA (amiRMADS27) that targets HvMADS27 mRNA. Root systems were analyzed using an Epson scanner and WinRHIZO software (Regent Instruments, Inc., Quebec, Canada). Total root length was further analyzed statistically using Statistica software 13.1 (Kraków, Poland). Densitometric analysis of western blots was performed using Image Studio Lite program version 5.2.5, and the amount of BG1 protein was normalized using WT control to normalize all bands (treated as 1.0, LI-COR, United States).
6
Radioactive Polymerase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IC50 values were determined in a similar polymerase activity assay as above but using radioactive labeling. The assay was performed in a total volume of 20 µL reaction mixture (Supplementary Table S2 ), but, as template, we used an RNA oligo containing a hairpin 5′-25U-HP-3′(5′-UUUUUUUUUUUUUUUUUUUUUUUUUAACAGGUUCUAGAACCUGUU-3′) and NTPs were replaced by 0.01 µCi/µL [α-32P]-ATP. After incubation, 5 µL of reaction mixtures was spotted on anion exchange cellulose filter paper (WhatmanTM Grade DE81 DEAE cellulose paper; GE Healthcare) in triplicate. The Whatman filter was then dried, subsequently washed by 0.125 mM Na2HPO4, water and ethanol and dried again. Dry filter paper was then analyzed using phosphorimaging. The plate was scanned on Amersham Typhoon 5 Biomolecular Imager (GE Healthcare), the products were quantified with Image Studio Lite (LI-COR) and data were analyzed using GraphPad version 6 (GraphPad Prism version 6, GraphPad Software, San Diego, CA, USA).
7
Quantitative Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The immunoprecipitated proteins from co-IP assays or ubiquitination assays or endogenous neuronal lysates were separated on reducing polyacrylamide gels and transferred to nitrocellulose membrane using standard procedures. The blots were probed with tag-specific or protein-specific primary antibodies followed by species-specific far-red conjugated secondary antibodies (Licor). Signal was detected using an Odyssey Imager (Licor). Western blots were analyzed using Li-Cor Image Studio Lite. Total fluorescence of labeled bands representing relative protein was measured. For all Western blot comparisons, the ratios of log transformed relative intensities (indicated for each blot) were analyzed using appropriate statistical tests as indicated in the corresponding figure legends.
8
Quantifying eIF4A1 Binding Kinetics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
25 nM Dy680- or Dy780-labelled RNAs were incubated with indicated proteins in AB + 2 mM AMPPNP/MgCl2 in the presence and absence of 100 μM silvestrol or 50 μM hippuristanol in 10 μl reactions for 60 min at 25°C.
In clamping experiments in Figure3H -I , eIF4A1 was preincubated with RNA and silvestrol in AB + 2 mM MgCl2 in the absence of nucleotide for 60 min at 25°C before competitor AG-RNA was added.
A final concentration of 2% (w/v) Ficoll-400 was added to the samples and complexes separated on 6–7% acrylamide-TB gels at 100 V for 50 min at room temperature using 0.5× TB as running buffer. When binding of eIF4A1 to the unwinding substrate was analysed, gels were run at 4°C. Gels were scanned immediately after the run with Odyssey (Licor) and band intensities quantified using Image Studio Lite. Dissociation constants were obtained from fitting the experimental data to the Hill-equation using Prism GraphPad 7, 8 or 9.
In clamping experiments in Figure
A final concentration of 2% (w/v) Ficoll-400 was added to the samples and complexes separated on 6–7% acrylamide-TB gels at 100 V for 50 min at room temperature using 0.5× TB as running buffer. When binding of eIF4A1 to the unwinding substrate was analysed, gels were run at 4°C. Gels were scanned immediately after the run with Odyssey (Licor) and band intensities quantified using Image Studio Lite. Dissociation constants were obtained from fitting the experimental data to the Hill-equation using Prism GraphPad 7, 8 or 9.
9
Western Blot Analysis of Astrocyte Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein from primary astrocyte cultures was isolated as previously described (Stary et al., 2017 (link)). Briefly, cultures were first washed with cold 0.1% phosphate buffered saline, then total cellular protein was quantified by Pierce BCA protein assay kit [ThermoFisher Scientific (Stary et al., 2017 (link))]. Equal amounts of protein were loaded and separated on 10–12.5% polyacrylamide gels, then transferred to Immobilon polyvinylidene fluoride membranes (EMD Millipore Corp). Membranes were blocked with 5% skimmed dry milk and incubated overnight with primary antibody against SIRT1 (Abcam, #ab110304), MFN2 (Abcam, #ab124773), β-actin (LI-COR Bioscience #926–42,210) and/or β-tubulin (Abcam, #ab6046). Membranes were then washed and incubated with secondary antibodies (LI-COR Bioscience) for 1 h followed by washing again and visualizing by using the LICOR Odyssey infrared imaging system. Densitometric analysis of bands was performed via Image Studio Lite (LI-COR Biosciences), and the intensity of all proteins was normalized to β-actin or β-tubulin as a control.
10
GALC-SapA Interaction Pulldown Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
mGALC was mixed with magnetic Ni-NTA agarose beads under saturating conditions (30 μl beads, 75 μg GALC per experiment) and incubated with mixing (90 min, 4 °C). Loaded beads were transferred to a flat bottomed 96-well plate and washed twice in 200 μl pulldown buffer containing 20 mM NaCl, 0.05% Tween-20 or LDAO and appropriate buffer. For the pH screen 100 mM phosphate buffer encompassing the pH range 4.5–7.5 was used while for subsequent pulldowns 100 mM sodium acetate pH 5.4 was used to more closely match that used in crystallisation experiments. Concentrated SapA was pre-incubated with 0.1% Tween-20 or LDAO for 1 hour and 160 µg of SapA was added to GALC-loaded beads in 200 μl pulldown buffer and incubated with shaking (2 hr, 4 °C). Beads were then washed for 60 s four times with 200 μl pulldown buffer. Proteins were eluted with 40 μl 500 mM imidazole, PBS pH 7.4, 0.05% Tween-20 and analysed by 4–12% gradient Bis-Tris PAGE. Following staining with Coomassie, gels were scanned on an Odyssey imaging system and band intensity determined using Image Studio Lite (LI-COR Biosciences). Uncropped gel images from pulldown experiments are provided in Supplementary Fig. 11 .
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!