Molecular imager versadoc mp 4000 system

Manufactured by Bio-Rad

Sourced in United States

The Molecular Imager VersaDoc MP 4000 System is a versatile imaging system designed for capturing and analyzing images of a variety of samples, including gels, blots, and microplates. The system features a high-resolution CCD camera, multiple illumination options, and software for image acquisition and analysis.

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Lab products found in correlation

Phosphatase inhibitor, by Roche (2 mentions) P7626, by Merck Group (1 mentions) A3687, by Merck Group (1 mentions) Bca1 1kt, by Merck Group (1 mentions) A4187, by Merck Group (1 mentions) P8340, by Merck Group (1 mentions) Image lab software, by Bio-Rad (1 mentions) Quantity one 1 d analysis software, by Bio-Rad (1 mentions) Supersignal west dura chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Lumi light western blotting substrate, by Roche (1 mentions) Quantity one software, by Bio-Rad (1 mentions) Ethidium bromide, by Merck Group (1 mentions) Hotstartaq master mix kit, by Qiagen (1 mentions) Millipore ultra free dna centrifugal filter, by Merck Group (1 mentions) Quantity one 1 d, by Bio-Rad (1 mentions) Enhanced chemiluminescence kit, by PerkinElmer (1 mentions) Ripa buffer, by Beyotime (1 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Roche (1 mentions) Bicinchoninic acid bca kit, by Beyotime (1 mentions) Immun star westernc chemiluminescence kit, by Bio-Rad (1 mentions)

12 protocols using molecular imager versadoc mp 4000 system

Quantifying Protein Expression in Corpus Luteum

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Fresh CL tissue (early CL, n = 6; mid CL, n = 6; late CL, n = 6) and in vitro tissue explants (mid CL, n = 6) were disrupted by homogenization in RIPA (250 μL) containing protease inhibitor (P8340, Sigma-Aldrich), phospho-stop solution (88667, ThermoFisher) and phenylmethylsulfonyl fluoride (PMSF) (P7626, Sigma-Aldrich) at 4°C. Protein concentration was determined with bicinchoninic acid assay (BCA) (BCA1-1KT, Sigma-Aldrich). A total of 10–80 μg of protein was run on 6–12% (varying accordingly to each protein) polyacrylamide gel followed by transfer to nitrocellulose membranes. Then, membranes were incubated with primary antibodies (Table 2) at 4°C, overnight. Goat anti-mouse alkaline phosphatase conjugated antibodies (1:30,000, 31321, ThermoFisher), goat anti-rabbit alkaline phosphatase-conjugated antibodies (1:30,000, A3687, Sigma-Aldrich), and rabbit anti-goat alkaline phosphatase-conjugated antibodies (1:30,000, A4187, Sigma-Aldrich) were used as a secondary antibody. Immune complexes were visualized using alkaline phosphatase substrate. Blots were scanned in Molecular Imager VersaDoc MP 4000 System (BioRad, Hercules, California, USA) and specific bands quantified using ImageLab Software (BioRad). At last, band density for each of the target protein was normalized against β-actin.

Walewska E., Wołodko K., Skarzynski D., Ferreira-Dias G, & Galvão A. (2019). The Interaction Between Nodal, Hypoxia-Inducible Factor 1 Alpha, and Thrombospondin 1 Promotes Luteolysis in Equine Corpus Luteum. Frontiers in Endocrinology, 10, 667.

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Colony Outgrowth Assay Protocol

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Colony outgrowth assays were performed as described previously (Fachinetti et al., 2017 (link); Niikura et al., 2010 (link)) with the following minor modifications. Cells (2.25 × 10⁵) were plated in a 6-well plate. The next day, 500 to 1000 μl of DMEM medium that contained retro-Cre virus (= filtered through a 0.45 μm membrane filter) was added to an equal volume of DMEM/F12 medium that contained 8 μg/ml polybrene (final 1:1 volume ratio of DMEM:DMEM/F12 medium). After 1 day, 500 or 5000 cells were plated in triplicate on a 6-well plate and cultured in DMEM/F12 medium. For 5000 or 500 cells’ plating, cells were selected with blasticidin S (10 μg/ml) 3–24 days or 3–28 days after virus infection of constructs, respectively. For 5000 or 500 cells’ plating, cells were grown 10 days or 14 days after the plating, respectively. After colony growth, colonies were fixed for 10 min in methanol and stained for 10 min in a crystal violet solution (2.3% crystal violet, 0.1% ammonium oxylate, 20% ethanol; Sigma-Aldrich). The number of colonies was counted with the Molecular Imager Versadoc MP4000 System (Bio-Rad), Quantity One 1-D Analysis Software (Bio-Rad), ImageJ software (NIH), and/or OpenCFU software.

Niikura Y., Kitagawa R., Fang L, & Kitagawa K. (2019). CENP-A Ubiquitylation Is Indispensable to Cell Viability. Developmental cell, 50(6), 683-689.e6.

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Western Blot Analysis of Cardiac Proteins

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EHMs were washed thoroughly in ice cold PBS and rapidly homogenized in lysis buffer containing 30 mmol/L Tris (pH 8.8), 5 mmol/L EDTA (pH 8), 3% SDS, 10% glycerol, 30 mmol/L NaF, and phosphatase inhibitors (Roche Applied Science). After centrifugation at 12,000 g for 10 min, supernatants containing soluble proteins were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. Primary antibodies used were: CSQ2 (1:2.500; ABR-01164 Dianova), GAPDH (1:50.000; RGM2-6CS Zytomed Systems), RyR2 (1:1.000; HPA020028 Sigma Aldrich Prestige), SERCA2a (1:200; sc-8094 Santa Cruz Biotech), LTCC (1:250; sc-81890 Santa Cruz). Primary antibodies were detected with appropriate horseradish peroxidase coupled secondary antibodies. Chemiluminescence signals were enhanced with the LumiLight Western Blotting Substrate (Roche) or SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific), detected with the Molecular Imager VersaDoc MP 4000 System (Bio-Rad), and quantified using QuantityOne software (Bio-Rad).

Godier-Furnémont A.F., Tiburcy M., Wagner E., Dewenter M., Lämmle S., El-Armouche A., Lehnart S.E., Vunjak-Novakovic G, & Zimmermann W.H. (2015). Physiologic force-frequency in engineered heart muscle by electromechanical stimulation. Biomaterials, 60, 82-91.

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Mink Insr Gene Expression Analysis

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We used the Basic Local Alignment Search Tool (BLAST) from the National Center of Biotechnology Information (NCBI: NIH http://www.ncbi.nlm.gov/sites/entrez?db=genome) to design PCR primers based on regions of homology of the Insr gene among rats, dogs, mice and humans. Using these primers, we amplified mink cDNA using Qiagen HotSTarTaq® Master Mix Kit (203443; QIAGEN). Each sample was subjected to 40 alternating cycles of a three-segment amplification program: 1) 30s denaturation at 95°C, 2) 1 min annealing at 57°C (beta actin) 59°C (Insr), 65°C (Gapdh), and 3) 1 min elongation at 72°C. The resulting cDNA fragments were placed in one percent Agarose gel containing 50ng/mL Ethidium Bromide (Sigma Aldrich, #E7637) and 1 x TAE (Tris-acetate-EDTA) buffer and subjected to electrophoresis at 60 volts for three hours and the resulting blots captured using Biorad’s Molecular Imager VersaDoc™ MP 4000 System (#170–8640). The bands containing the genes of interest were purified using a Millipore Ultra Free DNA Centrifugal Filter (42600; Millipore Corporation, Billerica, MA, USA). Based on the resulting nucleotide sequence for the Insr and housekeeping genes (Gapdh and Actb) primers were then designed specifically for mink (Table 1) and purchased from Integrated DNA Technologies (Coralville, IA, USA).

Hodonu A., Escobar M., Beach L., Hunt J, & Rose J. (2019). Glycogen metabolism in mink uterine epithelial cells and its regulation by estradiol, progesterone and insulin. Theriogenology, 130, 62-70.

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SDS-PAGE Protein Analysis Protocol

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All products of the time‐course reactions were stopped with 4xLaemmli sample loading buffer (Bio-Rad), resolved by 12% SDS–PAGE (in reducing or non-reducing conditions as indicated in the figures legends) and visualized by Alexa488 fluorescence emission in a Molecular Imager Versadoc MP4000 System (Bio‐Rad). Band densitometry was calculated by Quantity One 1-D (Bio-Rad).

Varejão N., Lascorz J., Codina-Fabra J., Bellí G., Borràs-Gas H., Torres-Rosell J, & Reverter D. (2021). Structural basis for the E3 ligase activity enhancement of yeast Nse2 by SUMO-interacting motifs. Nature Communications, 12, 7013.

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Roniciclib's Effects on Thyroid Cancer Cells

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Thyroid cancer cells were plated at 1 × 10⁶ cells in
100-mm Petri dishes in 10 mL of media overnight and treated with roniciclib at
25 nmol/L or vehicle for the indicated periods. Cell pellets were dissolved in
radio-immunoprecipitation assay buffer and protease inhibitor cocktail, vortexed
and clarified by centrifugation. Total protein (20–40 μg) was
separated by electrophoresis on 12% Tris-HCl gels, transferred to polyvinylidene
difluoride membranes, blocked and exposed to primary antibodies followed by a
secondary antibody conjugated to horseradish peroxidase. Signals were developed
using an enhanced chemiluminescence kit (PerkinElmer, Waltham, MA, USA). Band
density was imaged and quantified using Molecular Imager VersaDoc MP 4000 system
(Bio-Rad). The ratios of each protein to α-tubulin were calculated.

Lin S.F., Lin J.D., Hsueh C., Chou T.C, & Wong R.J. (2018). Potent effects of roniciclib alone and with sorafenib against well-differentiated thyroid cancer. Endocrine-related cancer, 25(10), 853-864.

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Mitochondrial Proteins Extraction and Analysis

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Frozen gastrocnemius muscle, liver and adipose tissues were lysed in RIPA buffer (Beyotime Biotechnology, Shanghai, China) supplemented with phosphatase inhibitors and PMSF (Roche, Ltd., Basel, Switzerland) before use. For the total MitoProfile Oxidative phosphorylation complexes (OXPHOS) analysis, mitochondrial proteins were extracted from the gastrocnemius muscle according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Protein concentrations were assayed using a bicinchoninic acid (BCA) kit (Beyotime Biotechnology, Shanghai, China). A total of 20–50 μg of protein were resolved on SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes. Information regarding the primary antibodies used in this study is provided in Table S1. Protein bands were visualized with Molecular Imager VersaDoc MP 4000 System (Bio-Rad, Berkeley, CA, USA).

Liu R., Li H., Fan W., Jin Q., Chao T., Wu Y., Huang J., Hao L, & Yang X. (2017). Leucine Supplementation Differently Modulates Branched-Chain Amino Acid Catabolism, Mitochondrial Function and Metabolic Profiles at the Different Stage of Insulin Resistance in Rats on High-Fat Diet. Nutrients, 9(6), 565.

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Gastric and Duodenal Tissue Protein Analysis

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Mouse gastric antrum and duodenal tissues (about 50 mg) were homogenized and lysed in 1 mL ice-cold RIPA buffer containing 2 mM PMSF (Beyotime, Shanghai, China). After centrifugation at 12,000 rpm for 10 min at 4 °C, the supernatant was collected and total protein concentration was determined by BCA Protein Assay Kit (CoWin Biosciences, Beijing, China). The protein was denatured by boiling for 5 min prior to undergoing 10% polyacrylamide gel electrophoresis, and was then transferred onto PVDF membranes (Bio-Rad, Hercules, CA). After blocking in 5% nonfat dry milk solution (Bio-Rad, Hercules, CA) for 2 h, the membranes were probed with primary antibodies against GAPDH (1:5000, Proteintech, Wuhan, China), C-kit (1:1000, CST, Boston, MA), 5-HT₄R (1:1000, Proteintech, Wuhan, China) overnight at 4 °C. Membranes were washed and incubated with HRP-conjugated anti-rabbit antibody (1:3000, CST, Boston, MA) for 2 h at room temperature. The protein was visualized using an Immun-Star WesternC Chemiluminescence Kit (Bio-Rad, Hercules, CA). The protein expression was observed using Molecular Imager VersaDoc MP 4000 System (Bio-Rad, Hercules, CA) and quantified by normalizing to GAPDH. Two-colour pre-dyed proteins (10–260 kDa, Bio-Rad, Hercules, CA) were used as molecular weight markers.

Gong Y., Liang X., Dai Y., Huang X., Su Q., Ma Y., Chen F, & Wang S. (2023). Prokinetic effects of Citrus reticulata and Citrus aurantium extract with/without Bupleurum chinense using multistress-induced delayed gastric emptying models. Pharmaceutical Biology, 61(1), 345-355.

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Quantitative Protein Analysis in Mice

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For protein analysis, three mice from each treatment group were randomly selected and harvested brain regions (e.g. cerebral cortical and cerebellar regions) were used for protein extraction and further analysis. The protein concentration of homogenized tissue samples [3:1 ratio of radioimmuno precipitation assay (RIPA) buffer with protease inhibitor and tissue sample] was determined by bicinchoninic acid (BCA) assay. Equal amounts of proteins were run on 10% SDS-PAGE followed by western blot analysis. The primary antibody for GLT-1 or GLAST was at 1:5000 dilution, and HRP-conjugated secondary antibody was used at 1:5000 dilution. All blots were developed using a Pierce chemiluminescence detection kit (Rockford, IL), followed by blot imaging and quantification with the Molecular Imager VersaDoc™ MP 4000 System and Image Lab Software version 5.2.1 from Bio-Rad, respectively.

Johnson J J.r., Pajarillo E.A., Taka E., Reams R., Son D.S., Aschner M, & Lee E. (2017). Valproate and sodium butyrate attenuate manganese-decreased locomotor activity and astrocytic glutamate transporters expression in mice. Neurotoxicology, 64, 230-239.

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Western Blot Analysis of EAG2 Protein

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For Western analysis, total proteins were extracted using a lysis buffer containing 50 mM Hepes (pH 7.4), 150 mM NaCl, 1% NP-40, 1 mM dithiothreitol (DTT), 1 mM EDTA supplemented with Complete Protease Inhibitor Cocktail (Roche). Protein lysate samples, 100 μg each, were resolved on 4–12% Bis-Tris gels using MOPS buffer (Invitrogen) and transferred onto a PVDF (Millipore) membrane using a buffer containing 25 mM Tris base, pH 8.3, 192 mM glycine, and 20% methanol for 1 hour at 100 V at 4°C. Western blot assays were performed using primary antibody diluted in TBS supplemented with 0.1% Tween-20 and 0.65% milk. Immunoreactive bands were visualized using horseradish peroxidase-conjugated secondary antibodies (Amersham), followed by chemiluminescence with ECL-plus Western Blotting Detection System (Amersham). Chemiluminescence was imaged and analyzed using Molecular Imager@VersaDoc MP4000 system (Bio-Rad). The primary antibodies used were: rabbit anti-EAG2 (Alomone, 1:1000), and mouse anti-αTubulin (Sigma, 1:10000).

Huang X., He Y., Dubuc A.M., Hashizume R., Zhang W., Reimand J., Yang H., Wang T.A., Stehbens S.J., Younger S., Barshow S., Zhu S., Cooper M.K., Peacock J., Ramaswamy V., Garzia L., Wu X., Remke M., Forester C.M., Kim C.C., Weiss W.A., James C.D., Shuman M.A., Bader G.D., Mueller S., Taylor M.D., Jan Y.N, & Jan L.Y. (2015). EAG2 potassium channel with evolutionarily conserved function as a brain tumor target. Nature neuroscience, 18(9), 1236-1246.

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