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Image studio version 3

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Image Studio version 3.1.4 is a software application developed by LI-COR for image analysis and quantification. It provides tools for processing and analyzing digital images, with a focus on western blot and fluorescence imaging applications.

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Lab products found in correlation

Odyssey clx imaging system, by LI COR (3 mentions) Odyssey imaging system, by LI COR (3 mentions) Prism 8, by GraphPad (3 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Reducing sample buffer, by Thermo Fisher Scientific (1 mentions) Actin hrp a3854, by Merck Group (1 mentions) Odyssey fc, by LI COR (1 mentions) Iblot 2 gel transfer device, by Thermo Fisher Scientific (1 mentions) Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Quick start bradford protein assay, by Bio-Rad (1 mentions) Odyssey clx imager system, by LI COR (1 mentions) Gnat1, by GeneTex (1 mentions) β tubulin, by Abcam (1 mentions) Odyssey blocking buffer, by LI COR (1 mentions) D phenylalanine, by Merck Group (1 mentions) Protease inhibitor cocktail, by Nacalai Tesque (1 mentions) Gapdh, by LI COR (1 mentions) Enhanced chemiluminescence kit, by Cytiva (1 mentions)

Close spelling variants of this product

Image Studio Lite version 3.1 Image Studio software (version 3.1). Image Studio software (version 3.1.4) Image Studio 3.1 software Image Studio

15 protocols using image studio version 3

1

Western Blot Analysis of Signaling Proteins

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Example 10

Lysis was performed using RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail (Roche). QuickStart Bradford protein assay (BioRad) was used to determine protein concentration with BSA as standard. Equal amounts of total protein were mixed with NuPAGE lithium dodecyl sulphate (LDS) and sample reducing buffer (Thermo Scientific), heated for 5 min at 95° C. and loaded into NuPAGE 10% or 12% Bis-Tris gels (Life Technologies) for electrophoresis. Separated proteins were transferred onto nitrocellulose membranes using iBlot 2 gel transfer device (Thermo Scientific). Blocking was performed with 5% milk or bovine serum albumin (BSA) in tris-buffered saline supplemented with 0.1% tween (TBST). AKT (4298S), ERK1/2 (4348S), p-AKT (9271S), and p-ERK (8544S) antibodies were from Cell Signaling Technology. RON antibody was made in-house and Actin-HRP (A3854) was from Sigma-Aldrich. Anti-rabbit (P0217) and anti-mouse (P0161) secondary antibodies were from Dako. The enhanced chemiluminescence (ECL) reagent used was SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, #34076). Imaging and acquisition was performed with Licor Odyssey Fc and Image Studio (version 3.1).

US11225515B2. Macrophage stimulating protein receptor (or RON—recepteur d'Origine Nantais) antibodies and uses thereof (2022-01-18). AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH [SG]. Inventors: David Lane [SG], Xin Yu Koh [SG], Le-Ann Hwang [SG].
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2

Thioesterase Activity Assay for RpfF

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The thioesterase reaction conditions were chosen based on previously published methodologies [15 (link),16 (link)]. RpfF thioesterase activity was measured using a 10-μL reaction consisting of 78 μM C12:0–ACPEc substrate, 0.64 μM RpfFBc (or control buffer N), and either 6.4 μM RpfRBc(FI), 1.28 μM RpfRBc(FI), or control buffer D in the thioesterase assay buffer (100 mM Tris [pH 7.5]). This reaction was incubated at 37 °C for 30 min and then heat inactivated at 95 °C for 2 min. Reactions were analyzed with a conformation-sensitive nondenaturing gel containing 20% polyacrylamide, 375 mM Tris (pH 8.8), and 2.5 M urea. Gels were stained with Coomassie Brilliant Blue, and band intensities were measured using a LI-COR Odyssey CLx Imager System (LI-COR Biosciences) and quantified using Image Studio version 3.1 (LI-COR Biosciences).
Waldron E.J., Snyder D., Fernandez N.L., Sileo E., Inoyama D., Freundlich J.S., Waters C.M., Cooper V.S, & Neiditch M.B. (2019). Structural basis of DSF recognition by its receptor RpfR and its regulatory interaction with the DSF synthase RpfF. PLoS Biology, 17(2), e3000123.
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3

Western Blot Analysis of Zebrafish Retinal Proteins

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Protein was extracted from freshly enucleated 2 mpf and 9 mpf zebrafish eyeballs with 150-300 μl of radio-immuno precipitation assay (RIPA) buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and protease inhibitor cocktail. 10 mg of total protein was separated on SDS-PAGE and transferred to PVDF membranes. After blocking with Odyssey Blocking Buffer (1:1 w/PBS; LI-COR Biosciences), the membrane was incubated with GNAT1 (GeneTex, Cat#GTX124622, 1:400 dilution), GNAT2 (1:500 dilution, MBL International, Cat#PM075), and β-tubulin (1:1000 dilution, Abcam, Cat#ab6046) antibodies in Odyssey Blocking Buffer (1:1 w/PBS+0.2%Tween 20) overnight at 4°C with gentle shaking. After washing with PBST (PBS with 0.1% Tween 20) three times, the membrane was incubated with IRDye 800cw or 680cw secondary antibodies in Odyssey Blocking Buffer (1:1 w/PBS+0.2% Tween 20+10% SDS; 1:20,000; LI-COR Biosciences) for 2 h at room temperature (RT). The membrane was then washed three times in PBST and once with PBS and was developed using an Odyssey CLx imaging system and Image Studio version 3.1 (LI-COR Biosciences). The pixel value of each band was measured using Image Studio Lite version 5.2. Quantitative analysis of the pixel value was performed using a two-tailed Student's t-test.
Yu M., Liu Y., Li J., Natale B.N., Cao S., Wang D., Amack J.D, & Hu H. (2016). Eyes shut homolog is required for maintaining the ciliary pocket and survival of photoreceptors in zebrafish. Biology Open, 5(11), 1662-1673.
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4

Quantitative Immunoblot Analysis

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All immunoblots were imaged using the Odyssey Imaging system and quantified using Image Studio version 3.1.4 (LI-COR). At least three independent experiments were performed for each analysis. Histogram bars represent mean ± SEM; P values: *<0.05; **<0.01; ***<0.001; ****< 0.0001, using unpaired Student t test.
Persaud A., Jiang C., Liu Z., Kefalas G., Demian W.L, & Rotin D. (2022). Elevated intracellular Na+ and osmolarity stimulate catalytic activity of the ubiquitin ligase Nedd4-2. Proceedings of the National Academy of Sciences of the United States of America, 119(30), e2122495119.
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5

Monitoring Autophagy Flux in LAPTM4b Knockdown

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HeLa cells stably knocked down for LAPTM4b (or control) were starved and treated with 50 μM chloroquine for the indicated times, lysed as described above and autophagic rate (flux) was monitored using LC3 antibodies. All blots were imaged using the Odyssey Imaging system and quantified using Image Studio version 3.1.4 (LI-COR). For autophagy immunofluorescent experiments, LAPTM4b stable knockdown and control cells were starved for 2 h in the presence of 50 μM chloroquine, fixed with 4% paraformaldehyde and permeabilized for 15 min with cold 95% methanol. Cells were blocked for 1 h with 1% BSA (in PBS) and incubated overnight in LC3 antibody (1:200) at 4 °C before being processed for imaging using Volocity, as above.
Milkereit R., Persaud A., Vanoaica L., Guetg A., Verrey F, & Rotin D. (2015). LAPTM4b recruits the LAT1-4F2hc Leu transporter to lysosomes and promotes mTORC1 activation. Nature Communications, 6, 7250.
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6

Regulation of mTORC1 by Amino Acids

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Cells were serum-starved overnight in RPMI 1,640 medium without amino acids and were restimulated with RPMI supplemented with EAA (or MEM) or 0.4 mM L-leucine (LEU222.25, Bioshop) for the indicated times. For knockdown of ATP6V0c experiment, HeLa cells were transfected with either control or ATP6V0c-715 siRNA (Abcam) and HA-LAPTM4b 48 h prior to serum starvation. For the ccA experiments, cells were transfected with HA-LAPTM4b where indicated and then treated with 5 μM ccA (Sigma) for 1 h prior to stimulation. For Rag A (Q66L) rescue experiments, HeLa cells were transfected with CFP-Rag A (Q66L) for 48 h, starved for 1.5 h and stimulated with EAA with or without 20 mM D-phenylalanine (P1751, Sigma) for the indicated times. Cells were lysed in lysis buffer and mTORC1 activation was monitored using anti-pT389-S6K1 and anti-p4E-BP1 antibodies. All blots were imaged using the Odyssey Imaging system and quantified using Image Studio version 3.1.4 (LI-COR).
Milkereit R., Persaud A., Vanoaica L., Guetg A., Verrey F, & Rotin D. (2015). LAPTM4b recruits the LAT1-4F2hc Leu transporter to lysosomes and promotes mTORC1 activation. Nature Communications, 6, 7250.
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7

LAT1 and 4F2hc Stability Assay

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HeLa cells (control KD, LAPTM4b KD or mCherry-LAPTM4b reconstituted into LAPTM4b KD cells) were treated with 50 μM cycloheximide for the indicated times. Cells were lysed and the stability of endogenous LAT1 and 4F2hc was monitored by immunoblotting. The relative abundance of each protein was normalized to actin. Images were quantified in Image Studio version 3.1.4 (LI-COR).
Milkereit R., Persaud A., Vanoaica L., Guetg A., Verrey F, & Rotin D. (2015). LAPTM4b recruits the LAT1-4F2hc Leu transporter to lysosomes and promotes mTORC1 activation. Nature Communications, 6, 7250.
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8

Comparative Statistical Analysis of Protein Expression

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Data are provided as means with SEM. Data were tested for normality with the Kolmogorov-Smirnov-Test, D’Agostino and Pearson omnibus normality test and Shapiro-Wilk-Test. Variances were tested using the Bartlettś test for equal variances. Data from repeated measurements were tested for significance using one-way ANOVA, and data from two groups were tested with paired or unpaired Student’s t-test, Mann-Whitney U-test using GraphPad Prism 8, GraphPad Software (San Diego, CA, www.graphpad.com). Densitometric analysis of western blots was done using Image Studio Version 3.1.4 (Licor). A p value <0.05 by two-tailed testing was considered statistically significant.
Xiao M., Bohnert B.N., Aypek H., Kretz O., Grahammer F., Aukschun U., Wörn M., Janessa A., Essigke D., Daniel C., Amann K., Huber T.B., Plow E.F., Birkenfeld A.L, & Artunc F. (2020). Plasminogen deficiency does not prevent sodium retention in a genetic mouse model of experimental nephrotic syndrome. Acta physiologica (Oxford, England), 231(1), e13512.
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9

Comprehensive Biostatistical Analysis Protocol

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Data are provided as means with SEM. Data were tested for normality with the Kolmogorov-Smirnov-Test, D'Agostino and Pearson omnibus normality test and Shapiro-Wilk-Test. Variances were tested using the Bartlett’s test for equal variances. Accordingly, data were tested for significance with parametric or nonparametric ANOVA followed by Bonferroni, Dunnett’s, Dunn’s, or Tukey's Multiple Comparison posthoc test, paired or unpaired Student’s t-test, or Mann-Whitney U-test where applicable using GraphPad Prism 8, GraphPad Software (San Diego, CA, www.graphpad.com). Densitometric analysis of Western blots was done using Image Studio Version 3.1.4 (Licor). A p value <0.05 at two-tailed testing was considered statistically significant.
Essigke D., Ilyaskin A.V., Wörn M., Bohnert B.N., Xiao M., Daniel C., Amann K., Birkenfeld A.L., Szabo R., Bugge T.H., Korbmacher C, & Artunc F. (2021). Zymogen-locked mutant prostasin (Prss8) leads to incomplete proteolytic activation of the epithelial sodium channel (ENaC) and severely compromises triamterene tolerance in mice. Acta physiologica (Oxford, England), 232(1), e13640.
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10

Statistical Analysis of Experimental Data

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Data are provided as means±s.e.m. Data were tested for normality with the Kolmogorov–Smirnov test, D'Agostino and Pearson omnibus normality test and Shapiro–Wilk test. Variances were tested using the Bartlett's test for equal variances. Accordingly, data were tested for significance with parametric or nonparametric ANOVA followed by Dunnett's, Dunn's or Tukey's multiple comparison post test, paired or unpaired Student's t-test, or Mann–Whitney U-test where applicable using Prism 8 (GraphPad Software, San Diego, CA, USA; www.graphpad.com). WB images were processed using Image Studio Version 3.1.4 (LI-COR). P<0.05 at two-tailed testing was considered statistically significant.
Bohnert B.N., Gonzalez-Menendez I., Dörffel T., Schneider J.C., Xiao M., Janessa A., Kalo M.Z., Fehrenbacher B., Schaller M., Casadei N., Amann K., Daniel C., Birkenfeld A.L., Grahammer F., Izem L., Plow E.F., Quintanilla-Martinez L, & Artunc F. (2021). Essential role of DNA-PKcs and plasminogen for the development of doxorubicin-induced glomerular injury in mice. Disease Models & Mechanisms, 14(9), dmm049038.
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