Las 3000

Manufactured by GE Healthcare

Sourced in United States, Japan

The LAS-3000 is a laboratory imaging system designed for the analysis and documentation of biomolecular samples. The device utilizes luminescence detection technology to capture and analyze images of samples, such as Western blots, DNA gels, and protein arrays. The LAS-3000 provides high-resolution imaging capabilities and can be used in a variety of life science research applications.

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Lab products found in correlation

Ecl prime, by GE Healthcare (4 mentions) Bca protein assay kit, by Thermo Fisher Scientific (3 mentions) Ecl kit, by GE Healthcare (2 mentions) Sc 7963, by Santa Cruz Biotechnology (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Las4000, by GE Healthcare (2 mentions) Ecl prime immunoblotting detection kit, by GE Healthcare (2 mentions) Protease inhibitor, by Roche (2 mentions) Pvdf membrane, by Bio-Rad (2 mentions) Phosstop, by Roche (2 mentions) Pvdf membrane, by GE Healthcare (2 mentions) Nitrocellulose membrane, by Cytiva (2 mentions) Superscript 3, by Thermo Fisher Scientific (1 mentions) Sybr green 1, by Takara Bio (1 mentions) Gotaq polymerase, by Promega (1 mentions) Edta free protease and phosphatase inhibitor cocktails, by Roche (1 mentions) T per, by Thermo Fisher Scientific (1 mentions) Protein assay, by Bio-Rad (1 mentions) Protease and phosphatase inhibitors, by Nacalai Tesque (1 mentions) Enhanced chemiluminescence, by GE Healthcare (1 mentions)

36 protocols using las 3000

Quantitative RT-PCR Analysis of P19 Cells

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The total RNAs of P19 cells were prepared as described above. The cDNAs were produced from 2 μg of total RNAs using SuperScript III (Life technologies) and 0.5 μg of oligo dT primer in a 20 μL reaction mixture. The cDNAs are used as templates for PCR. GoTaq polymerase (Promega, Fitchburg, WI, USA) with specific primers performed the PCR reactions. The DNA primer sequences, number of cycles and annealing temperatures for the candidates are described in Additional file 1: Table S1. Primers and conditions for β-Actin and GluR1 (controls) were described in a previous report [34 (link)]. The PCR products were analyzed on a 6% polyacrylamide gel. The gels were stained with SYBR Green I (Takara Bio Inc., Otsu, Japan), and a LAS-3000 (GE Healthcare, Fairfield, CT, USA) was used to analyze the images. The sequences of the PCR products were confirmed in a 3100 DNA sequencer (Life technologies). MultiGauge v 3.0 software (GE Healthcare) was used to perform the densitometry. Each experiment was performed at least three times to confirm reproducibility.
As for Ethics, this research did not involve any human subject, human material, or human data, and was not performed on any animals. This research involved Recombinant DNA Experiments and was approved by Life science committee of Japan Advanced Institute of Science and Technology.

Alam S., Phan H.T., Okazaki M., Takagi M., Kawahara K., Tsukahara T, & Suzuki H. (2014). Computational extraction of a neural molecular network through alternative splicing. BMC Research Notes, 7, 934.

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Protein Extraction and Western Blotting of Postmortem Tissues

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Human postmortem tissue was homogenized using Tissue Protein Extraction Reagent (T-PER, 0.1 g/mL, Thermo Scientific, Waltham, USA) containing EDTA-free protease and phosphatase inhibitor cocktails (1:25 and 1:10 respectively, Roche, Basel, Germany). Samples were centrifuged at 10,000 g for 15 min at 4°C. Protein content in the supernatant was quantified using Bio-Rad protein assay (Bio-rad, Hercules, USA). Human postmortem homogenates (15 μg) were prepared in samples buffer (2% SDS, 0.03 M Tris, 5% 2-Mercaptoethanol, 10% glycerol, bromophenol blue) and heated 5 min at 95°C. Electrophoresis of postmortem samples was carried out using pre-cast NuPAGE Bis-Tris Mini Gels 4–12% (1.5 mm, 4–12%; Invitrogen, Carlsbad, USA). After blotting, the membranes were blocked in 5% milk/PBS for 1 h, followed by incubation with primary antibody in 5% milk, 0.1% Tween/PBS for 1 h at RT. The membrane was washed three times in 0.1% Tween/PBS and secondary antibodies in 5% milk, 0.1% Tween/PBS were added for 1 h at RT. After washing, enhanced chemoluminescence detection reagent (GE Healthcare) was added according to the manufacturer protocol and images were acquired using a CCD camera (LAS-3000).

Dolfe L., Tambaro S., Tigro H., Del Campo M., Hoozemans J.J., Wiehager B., Graff C., Winblad B., Ankarcrona M., Kaldmäe M., Teunissen C.E., Rönnbäck A., Johansson J, & Presto J. (2018). The Bri2 and Bri3 BRICHOS Domains Interact Differently with Aβ42 and Alzheimer Amyloid Plaques. Journal of Alzheimer's Disease Reports, 2(1), 27-39.

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Phosphorylation Analysis in Cancer Cells

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For analyzing the phosphorylation/activation pattern of relevant molecules, cancer cells were treated with YHO-1701 and/or imatinib, osimertinib, or alectinib for 24 h; following this, they were lysed using RIPA buffer containing protease and phosphatase inhibitors (Nacalai Tesque, Inc., Kyoto, Japan). Lysate concentrations were determined using the BCA protein assay kit (Pierce Chemical Co., Rockford, IL) and normalized for protein load. The lysate was then separated on SDS–polyacrylamide gel electrophoresis gels and transferred onto polyvinylidene fluoride membranes. The membrane was treated with primary antibodies overnight at 4 °C. Proteins were detected using horseradish peroxidase-conjugated secondary antibodies, visualized with an ECL kit (GE Healthcare Biosciences), and exposed to LAS-3000 (GE Healthcare Biosciences).

Taniguchi K., Konishi H., Yoshinaga A., Tsugane M., Takahashi H., Nishisaka F., Shishido Y, & Asai A. (2021). Efficacy of combination treatment using YHO-1701, an orally active STAT3 inhibitor, with molecular-targeted agents on cancer cell lines. Scientific Reports, 11, 6685.

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Western Blot Analysis of β-catenin and CXXC5

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Cells and tissues were lysed using radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0% Triton X‐100, 1% sodium deoxycholate and 5 mM EDTA). Samples were separated on 12% SDS polyacrylamide gels and transferred onto PROTRAN nitrocellulose membranes (Shleicher and Schuell, Co., NH). After blocking with PBS containing 5% non‐fat dry skim milk and 0.07% Tween 20, the membranes were incubated with antibodies specific for β‐catenin (1:1000, sc‐7963, Santa Cruz Biotechnology, Inc.), CXXC5 (1:500, Lab made) and β‐actin (1:1000, ab8226, Abcam) at 4°C for 12 h. Membranes were then incubated with horseradish peroxidase‐conjugated anti‐rabbit (Bio‐Rad, CA) or anti‐mouse (Cell Signaling Technology) IgG secondary antibody. Protein bands were visualized with enhanced chemiluminescence (GE Healthcare, IL) using a luminescent image analyser (LAS‐3000).

Seo S.H., Kim E., Lee S., Lee Y., Han D.H., Go H., Seong J.K, & Choi K. (2022). Inhibition of CXXC5 function reverses obesity‐related metabolic diseases. Clinical and Translational Medicine, 12(4), e742.

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Protein Extraction and Western Blot Analysis

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Extraction of proteins from whole-cell lysates was performed as described previously^{12 (link), 13 (link)}. Extraction of proteins from conditioned culture medium was performed as follows: Cells (5 × 10⁶) were cultured in RPMI-1640 medium without fetal bovine serum (FBS) for 24 h. Conditioned medium was harvested and added to trichloroacetic acid at 20% of the final concentration. This sample was incubated on ice for 20 min and centrifuged at 15,000 × g for 5 min. The pellet was washed in cold acetone and then dissolved in lysis buffer containing a protease inhibitor cocktail (Roche, Mannheim, Germany).
WB analysis was performed as previously described^{12 (link), 13 (link)}. Briefly, 30 μg of protein was transferred onto the membrane. The membrane was incubated with primary antibodies overnight at 4 °C and then with secondary antibodies for 30 min. Immune complexes were detected using ECL Plus (GE Healthcare, Piscataway, NJ, USA) and an LAS-3000 (GE Healthcare). β-actin served as an internal control.

Baba K., Kitajima Y., Miyake S., Nakamura J., Wakiyama K., Sato H., Okuyama K., Kitagawa H., Tanaka T., Hiraki M., Yanagihara K, & Noshiro H. (2017). Hypoxia-induced ANGPTL4 sustains tumour growth and anoikis resistance through different mechanisms in scirrhous gastric cancer cell lines. Scientific Reports, 7, 11127.

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Comprehensive Western Blot Analysis

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Western blot analysis was performed as previously described^{17 (link)}. Cells were lysed using radio-immunoprecipitation assay (RIPA) buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0% Triton X-100, 1% sodium deoxycholate, and 5 mM EDTA). Samples were separated on 12% SDS polyacrylamide gels and transferred onto PROTRAN nitrocellulose membranes (Shleicher and Schuell Co.). After blocking with PBS containing 5% nonfat dry skim milk and 0.07% (vol/vol) Tween 20, the membranes were incubated with antibody specific for β-catenin (1:1000, sc-7963, Santa Cruz Biotechnology, Inc.), CXXC5 (1:500, Lab made), PPARγ (1:1000, ab19481, Abcam), or C/EBPα (1:1000, 2295, Cell Signaling Technology) at 4 °C overnight. Membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit (1:1000, 170-6515, Bio-Rad) or anti-mouse (1:1000, 14790, Cell Signaling Technology) IgG secondary antibody. Protein bands were visualized with enhanced chemiluminescence (GE Healthcare) using a luminescent image analyzer, LAS-3000.

Seo S.H., Lee D., Lee S.H, & Choi K.Y. (2022). Blockade of CXXC5-dishevelled interaction inhibits adipogenic differentiation, obesity, and insulin resistance in mice. Scientific Reports, 12, 20669.

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Protein Detection Using SDS-PAGE and PVDF Blotting

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Samples were run on SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF)
membrane (Millipore) by using a semi-dry transfer cell (Trans blot SD, Bio-Rad
laboratories Inc., Hercules, CA, USA). The membrane was then blocked with 5% skim milk in
Tris buffer saline (TBS; 25 mM Tris-HCl, [pH 7.5],0.14 M NaCl) at room temperature for 30
min followed by treatment with affinity-purified rabbit anti-NetB IgG (5 µg/ml) or rabbit
anti-caveolin-1 polyclonal IgG (ECM Biosciences, Versailles, KY, USA) (1:2,000) at room
temperature for 1 hr. The membrane was washed with TBST (0.05% Tween 20 in TBS) and
incubated with 3,000-times diluted peroxidase-labeled goat anti-rabbit IgG (GE Healthcare)
at room temperature for 30 min. Detection was performed using a chemiluminescence kit
(Super Signal^® West Femto Maximum Sensitivity Substrate; Thermo Fisher
Scientific Inc.). The method was based on the manual attached to the kit. For observation
and photographing of chemiluminescence, a lumino image analyzer LAS-3000 (GE Healthcare)
was used.

ISLAM A.A., NAKATANI M., NAKAJIMA T., KOHDA T, & MUKAMOTO M. (2020). The cytotoxicity and molecular mechanisms of the Clostridium perfringens NetB toxin. The Journal of Veterinary Medical Science, 83(2), 187-194.

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Immunoblot Analysis of Rice Grain Amylase

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The immunoblot analysis was performed as described previously^{24 (link)}. Crude protein samples from mature grains of rice were separated on a 12% SDS-PAGE gel and blotted onto a PVDF membrane. The membrane was blocked in PBST buffer (8.1 mM Na₂HPO₄, 137 mM NaCl, 1.5 mM KH₂PO₄, 2.7 mM KCl and 0.1% Tween-20, pH7.5) with 5% fat-free milk 25 °C for 1 h. Blots were incubated with specific antibodies detecting the amylase isoforms^{30 (link)} at 25 °C for 1 h, and then with horseradish peroxidase (HRP)-linked anti-rabbit immunoglobulin G. Bands were quantitatively detected using chemiluminescent HRP substrate ECL Prime (Amersham) and the chemiluminescent analyzer, LAS3000 (GE Healthcare). Signals were normalized by the intensity of Coomassie brilliant blue staining with the corresponding protein.

Sera Y., Hanamata S., Sakamoto S., Ono S., Kaneko K., Mitsui Y., Koyano T., Fujita N., Sasou A., Masumura T., Saji H., Nonomura K.I., Mitsuda N., Mitsui T., Kurusu T, & Kuchitsu K. (2019). Essential roles of autophagy in metabolic regulation in endosperm development during rice seed maturation. Scientific Reports, 9, 18544.

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Quantifying HP1 Binding to 601 DNA

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Various concentrations of HP1 proteins were incubated with 0.2 pmol of 601 DNA in 10 μl of EMSA assay buffer containing 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM DTT and 0.1 mg/ml BSA for 15 min at 37°C. After incubation, 1 μl of 30% sucrose was added, and the samples were loaded onto 5% native polyacrylamide gels in 0.5× Tris-borate-EDTA. Gels were run at room temperature at 100 V for 1.5 h. Gels were stained with SYBR Gold (Invitrogen), visualized using a LAS3000 (GE Healthcare) and quantified using ImageQuant software. Binding curves were fitted with the following equation: fraction bound = 1 − fraction unbound. Curve-fitting was performed with Igor Pro software.

Nishibuchi G., Machida S., Osakabe A., Murakoshi H., Hiragami-Hamada K., Nakagawa R., Fischle W., Nishimura Y., Kurumizaka H., Tagami H, & Nakayama J.I. (2014). N-terminal phosphorylation of HP1α increases its nucleosome-binding specificity. Nucleic Acids Research, 42(20), 12498-12511.

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Comprehensive Protein Expression Analysis

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The detailed protocols of the total cell lysate extraction, Western blot analysis, and IHC have been described previously.11, 12 The primary antibodies were as follows: p‐HER2 (Tyr1221/1222), HER2, p‐EGFR (Tyr1068), EGFR, p‐MAPK (Erk1/2) (Thr202/Tyr204), MAPK (Erk1/2), p‐AKT (Ser473), AKT, cleaved PARP (Asp214), IGF‐I receptor β (IGF‐1R), p‐IGF‐I receptor β (phospho‐IGF‐1R) (Tyr1135/1136) (Cell Signaling Technology), and actin (Santa Cruz Biotechnology). The following secondary antibodies were used in the Western blotting: anti‐rabbit, anti‐mouse (Santa Cruz Biotechnology). To detect specific proteins, the membranes were examined using the ECL Prime Western Blotting Detection System (GE Healthcare) and LAS‐3000 (Fuji‐film). As for IHC staining, the clinical tumors were stained using anti‐HER2 primary antibody purchased from Roche Diagnostics.

Yoshioka T., Shien K., Namba K., Torigoe H., Sato H., Tomida S., Yamamoto H., Asano H., Soh J., Tsukuda K., Nagasaka T., Fujiwara T, & Toyooka S. (2018). Antitumor activity of pan‐HER inhibitors in HER2‐positive gastric cancer. Cancer Science, 109(4), 1166-1176.

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