Las4000 luminescence imager

Manufactured by Fujifilm

Sourced in Japan

The LAS4000 is a luminescence imager designed for the detection and analysis of luminescent signals. It features a high-resolution CCD camera and advanced imaging optics to capture detailed images of luminescent samples. The LAS4000 is capable of detecting a wide range of luminescent signals, including chemiluminescence, bioluminescence, and fluorescence, making it a versatile tool for various applications in life science research and analysis.

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8 protocols using las4000 luminescence imager

Transgene Copy Number Determination

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100 mg fresh leaves transgenics were homogenized in liquid nitrogen and mixed with 500 μl CTAB buffer (2% w/v CTAB, 0.3 M NaCl, 20 mM EDTA, 100 mM Tris–Cl (pH-8) 0.2% BME). Genomic DNA was isolated according to Murray and Thompson³⁶. Quality and quantity of genomic DNA was checked by gel electrophoresis and nanodrop (GE, US), respectively.
Transgene copy number was checked using 10 μg of genomic DNA. DNA was digested with BamHI and separated by 0.8% agarose gel electrophoresis. DNA was blotted on Hybond-N Nylon membrane (Pharmacia) and fixed using UV cross linker (UVP HybriLinker, Analytik Jena, US). Blot was probed with hpt gene probe which was synthesized using PCR DIG probe synthesis kit (SIGMA-ALDRICH). Hybridization and detection were done using the non-radioactive method by DIG Luminescent Detection Kit as per the manufacturer guidelines (SIGMA-ALDRICH) with hybridization temperature 42 °C and washing 65 °C. Detection was carried out using CSPD solution and visualized by Fujifilm LAS4000 luminescence imager.

Sharma N., Chaudhary C, & Khurana P. (2020). Wheat Myo-inositol phosphate synthase influences plant growth and stress responses via ethylene mediated signaling. Scientific Reports, 10, 10766.

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Characterizing Oxidized DJ-1 Protein

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Protein
samples were denatured in the SDS sample buffer [63 mM Tris-HCl (pH
6.8) containing 0.012% bromophenol blue, 5% sucrose, and 2% SDS] for
5 min at 95 °C (nonreduced condition). To reduce protein samples,
1% beta-mercaptoethanol (2-ME) was added to SDS sample buffer and
then heat treatment was conducted. After separation of the samples
by SDS-PAGE (12.5% gel), the proteins were transferred to an Immobilon-P
Transfer Membrane (Millipore, Bedford, MA) for the western blot analysis.
The membranes were blocked in a Tris-buffered saline (pH 7.4) containing
0.1% Tween 20 (TBS-T) containing 5% skimmed milk powder (Snow Brand
Milk Products, Tokyo, Japan), incubated with anti-oxDJ-1 mAb (clone
M106, 1 μg/mL, ref (18 (link))), mouse anti-DJ-1 mAb (clone 3E8, 1 μg/mL, Medical
& Biological Laboratories, Nagoya, Japan), or anti-β-actin
mAb (AC-15) at 4 °C for 18 h, and incubated with HRP-conjugated
secondary antibodies for at least 1 h. After the incubation with Immobilon
Western (Millipore), the immunoreactivity was visualized with an LAS-4000
luminescence imager (Fujifilm, Tokyo, Japan). For silver staining,
the separated proteins were stained with the Dodeca Silver Stain Kit
(Bio-Rad Laboratories, Hercules, CA).

Kobayashi M., Muramatsu K., Haruyama T., Uesugi H., Kikuchi A., Konno H., Noguchi N, & Saito Y. (2019). Polymerization of Oxidized DJ-1 via Noncovalent and Covalent Binding: Significance of Disulfide Bond Formation. ACS Omega, 4(6), 9603-9614.

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Western Blot Protein Detection

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Cells were washed once with PBS and lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche). Protein concentration was determined using a BCA kit (Pierce). Equal amounts of lysate were separated by SDS-PAGE and western blot analysis conducted using the antibodies indicated above. Primary antibodies were detected with HRP-conjugated secondary antibody (Santa Cruz Biotechnology) and detected with Western Lightning (Perkin Elmer) or Immobilon (Millipore) chemiluminescent HRP substrate. Blots were imaged using an LAS 4000 luminescence imager (Fujifilm). Densitometry was determined using Image J software (NIH).

, & Massey A.J. (2017). Modification of tumour cell metabolism modulates sensitivity to Chk1 inhibitor-induced DNA damage. Scientific Reports, 7, 40778.

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Western Blot Analysis of Protein Lysates

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Following washing with PBS, cells were lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche). Protein concentration was determined using a BCA kit (Pierce). Equal amounts of lysate were separated by SDS-PAGE and western blot analysis conducted using the antibodies indicated above. Primary antibodies were detected with HRP-conjugated secondary antibody (Santa Cruz Biotechnology) and detected with Western Lightning (Perkin Elmer) or Immobilon (Millipore) chemiluminescent HRP substrate. Blots were imaged using an LAS 4000 luminescence imager (Fujifilm). Densitometry was determined using Image J software (NIH).

, & Massey A.J. (2016). Tumour growth environment modulates Chk1 signalling pathways and Chk1 inhibitor sensitivity. Scientific Reports, 6, 35874.

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Investigating TaMIPS-B Protein Interactions

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To check the TaMIPS-B protein interaction, Far-western analysis was carried out using purified TaMIPS-B-His recombinant protein as bait and plant crude extract (Zadok 65 spike stage) as prey according to Wu et al.^{42 (link)}. 500 μg prey protein along with BSA (Bovine serum albumin) and purified His-TaMIPS-B as negative and positive control, respectively, were blotted on membrane in two sets and one of it was proceeded with western blot analysis and the other blot used for far-western analysis. Western blot was probed and detected by anti-His antibody whereas other was proceeded to denaturing/renaturing of blotted prey protein for 12–16 h. Blot was then blocked with 5% milk and washed with PBST (Phosphate buffered saline with Tween-20) buffer three times. Membrane was then incubated with bait protein (Purified His-TaMIPS-B protein) at 4 °C for 12 h. Bait protein bound to prey protein was detected on blot after stringent washing and incubating it with primary (anti-His) antibody followed by secondary antibody. Visualization was done by chemiluminescence using Immobilon Western Chemiluminescent HRP Substrate (MERCK) according to manufactural instructions using Fujifilm LAS4000 luminescence imager.

Sharma N., Chaudhary C, & Khurana P. (2020). Wheat Myo-inositol phosphate synthase influences plant growth and stress responses via ethylene mediated signaling. Scientific Reports, 10, 10766.

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Chromatin Immunoblot Analysis of Histone Modifications

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Seeds were surface sterilized in 70% ethanol 0.05% SDS for 3 min and rinsed with 90% ethanol before drying and plating on MS medium supplemented with 0.5% sucrose and 0.9% agar. Eight-day-old seedlings were used for chromatin extraction protocol as described previously [101 (link)]. Forty micrograms of protein samples, as estimated by by Pierce BCA Protein Assay Kit (Thermo Scientific), was loaded on 14% LiDs Tris-Tricine gels and blotted onto Immobilon-P membranes (Millipore) before immunodetection using antibodies recognizing either unmodified histone H4 (Millipore #05-858) or H3K27me3 (Millipore #07-449), H3K4me3 (Millipore #07-745R), and H3K9ac (Millipore 06-942, lot: 31636). Chemiluminescent signals were detected and quantified using a LAS4000 luminescence imager (Fuji) in three biological replicates.

Rutowicz K., Lirski M., Mermaz B., Teano G., Schubert J., Mestiri I., Kroteń M.A., Fabrice T.N., Fritz S., Grob S., Ringli C., Cherkezyan L., Barneche F., Jerzmanowski A, & Baroux C. (2019). Linker histones are fine-scale chromatin architects modulating developmental decisions in Arabidopsis. Genome Biology, 20, 157.

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Western Blot Protein Detection Protocol

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Cells were washed twice with phosphate-buffered saline (PBS), and lysed in buffer containing 137 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl (pH 7.4), 10% glycerin, 1% Triton X-100 and a protein inhibitor cocktail (Roche Diagnostics) for 30 min on ice. The lysate was centrifuged at 14,000

\times

g for 20 min at 4

^{\circ}

C, and the supernatants were collected. Samples were subjected to SDS-PAGE in a 10% acrylamide gel and the resolved protein bands were transferred electrophoretically onto a nitrocellulose membrane. The membranes were blocked for 1.5 h at room temperature in Tris-buffered saline (TBS) containing 5% nonfat dry milk and 0.1% Tween-20, after which the membranes were probed with primary antibodies at 4

^{\circ}

C overnight. After washing in TBS, the membranes were incubated in TBS containing horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Sigma-Aldrich). Bound primary antibody was visualized using an enhanced chemiluminescence (ECL) detection kit (GE Healthcare, Chicago, IL, USA), and the membranes were scanned using an LAS-4000 luminescence imager (Fujifilm, Tokyo, Japan). After stripping the membranes in 20 mM glycine-HCl (pH 2.3), protein loading normalization was performed by probing with anti-

β

-actin antibody, incubation with HRP-anti-mouse antibody and visualized using ECL.

Liu Z., Ma M., Yan L., Chen S., Li S., Yang D., Wang X., Xiao H., Deng H., Zhu H., Zuo C, & Xia M. (2018). miR-370 regulates ISG15 expression and influences IFN- sensitivity in hepatocellular carcinoma cells. Cancer Biomarkers, 22(3), 453-466.

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Chromatin Extract Immunodetection Protocol

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Soluble protein samples were obtained using the indicated methods, and chromatin extracts were obtained as previously described [60 (link)]. Unless stated, 10 μg of protein samples were loaded on 14% LiDS Tris-Tricine gels and blotted onto PVDF membranes before immunodetection and analysis using a LAS4000 luminescence imager (Fuji). The following antibodies were used: anti-H3 (Millipore #07-690), anti-H3K4me1 (Active Motifs #39297), anti-H3K4me2 (Millipore #07-030), anti-H3K4me3 (Millipore #05-745), anti-GFP (Clontech #632381), anti-MYC antibodies (Millipore #05-724), or custom-designed anti-rice histone H2B [60 (link)]. Anti-WDR5 serum was obtained by immunization of a rabbit with a 50-amino-acid synthetic peptide corresponding to amino acids 42–91 of the Arabidopsis WDR5a protein and affinity purification by the SDIX company (USA). All uncropped blots are given in Additional file 12.

Fiorucci A.S., Bourbousse C., Concia L., Rougée M., Deton-Cabanillas A.F., Zabulon G., Layat E., Latrasse D., Kim S.K., Chaumont N., Lombard B., Stroebel D., Lemoine S., Mohammad A., Blugeon C., Loew D., Bailly C., Bowler C., Benhamed M, & Barneche F. (2019). Arabidopsis S2Lb links AtCOMPASS-like and SDG2 activity in H3K4me3 independently from histone H2B monoubiquitination. Genome Biology, 20, 100.

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