Ls reloaded

Manufactured by Tecan

Sourced in Switzerland

The LS Reloaded is a liquid handling system designed for automated sample preparation and liquid handling tasks in life science laboratories. It offers precise and reliable liquid handling capabilities to support a wide range of applications.

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Lab products found in correlation

α flag cy3, by Merck Group (2 mentions) Proteinase k, by Thermo Fisher Scientific (2 mentions) Anti c myc cy3, by Merck Group (1 mentions) Genepix 7, by Molecular Devices (1 mentions)

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LS Reloaded scanner

11 protocols using ls reloaded

Microarray-based Cytokine Detection Assay

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Each standard curve consisted of 12 points spanning the full range of the assay, including an assay blank of 0.1% casein in PBS. Toxins were diluted into the appropriate sample diluent. Twenty microliters of diluted toxin was applied to each well of the microarray and incubated for up to 16 hours on a gently rotating orbital shaker. Three washes were performed after each incubation step with PBS containing 0.05% Tween 20 (PBS-T). The slides were then incubated with the appropriate mix of biotinylated detection antibodies and incubated for 2 hours. The signal was enhanced using the biotinyltyramide amplification system.^{54 (link),55 (link)} The slides were incubated with 1 μg mL⁻¹ HRP-conjugated streptavidin for 30 minutes, followed by incubation for 10 minutes with 1 μg mL⁻¹ biotinyltyramide. Finally, the slides were incubated with 1 μg mL⁻¹ Cy3-conjugated streptavidin for 30–60 minutes in the dark with gentle rocking followed by a final wash and then rinsed with distilled water and dried. Experiments in the shortened assay series were incubated as indicated in the text. Cy3 fluorescence was detected by scanning slides on an LS Reloaded (Tecan, Switzerland) microarray scanner (laser: 532 nm; filter: 575 nm).

Jenko K.L., Zhang Y., Kostenko Y., Fan Y., Garcia-Rodriguez C., Lou J., Marks J.D, & Varnum S.M. (2014). Development of an ELISA microarray assay for the sensitive and simultaneous detection of ten biodefense toxins. The Analyst, 139(20), 5093-5102.

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U2B'' and U2A’ protein expression and immobilization

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The template for N' terminal cMYC, and C' terminal 6 x HIS U2B" (Tb927.3.3480) or U2A’ (Tb927.10.14360) synthetic gene was created by assembly PCR (26 (link)) using primers listed in Supplementary Table ST1. U2B" and U2A’ protein containing 5′-cMyc and 3′-HIS tag was synthesized using rabbit reticulocyte quick coupled transcription and translation system (Promega). The 5′-cMyc and 3′-HIS tagged U2B" and U2A’ protein was then immobilized underneath the MITOMI valve using anti HIS-biotin (33 μg/ml, QIAGEN) and detected using anti cMyc-Cy3 (13 μg/ml, Sigma-Aldrich) as previously described (26 (link)). The fluorescent signal intensities were measured using a microarray laser scanner (LS Reloaded, TECAN) and the data were extracted using GenepixPro software.

Rajan K.S., Doniger T., Cohen-Chalamish S., Chen D., Semo O., Aryal S., Glick Saar E., Chikne V., Gerber D., Unger R., Tschudi C, & Michaeli S. (2019). Pseudouridines on Trypanosoma brucei spliceosomal small nuclear RNAs and their implication for RNA and protein interactions. Nucleic Acids Research, 47(14), 7633-7647.

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Protein-Protein Interaction Assay on Chip

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Protein-protein interactions on chip were carried out as previously described (Gerber et al., 2009 (link)). Human proteins (C-TIP, PPARα and HIF1α) with N terminus c-Myc and C terminus His tags were expressed by using rabbit reticulocyte quick-coupled TNT reaction (Promega). Surface chemistry was prepared and α-HIS biotinylated antibodies (QIAGEN) were bound under buttons on chip. Next, each expressed protein was attached to a specific part of the device and bound to its corresponding antibody. Finally, Flag-tagged SIRT6 was applied to the device. By closing the sandwich valves, each unit cell separated from its environment. SIRT6 were allowed to incubate with the proteins for 30 min at 32°C. Next, the button valves opened, exposing proteins, and unbound protein was washed away. Bound proteins were then labeled with α-c-Myc Alexa Fluor 647 (Abcam) and α-FLAG cy3 (Sigma) antibodies. Protein interactions were determined with a microarray scanner (LS Reloaded; Tecan) using a 633-nm laser and 695-nm filter for Alexa Fluor 647 and a 535-nm laser and 595-nm filter for Cy3.

Naiman S., Huynh F.K., Gil R., Glick Y., Shahar Y., Touitou N., Nahum L., Avivi M.Y., Roichman A., Kanfi Y., Gertler A.A., Doniger T., Ilkayeva O.R., Abramovich I., Yaron O., Lerrer B., Gottlieb E., Harris R.A., Gerber D., Hirschey M.D, & Cohen H.Y. (2019). SIRT6 Promotes Hepatic Beta-Oxidation via Activation of PPARα. Cell reports, 29(12), 4127-4143.e8.

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Quantifying On-Chip DNA Concentration

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To determine the actual on-chip printed DNA concentration, Cy5-labeled oligos with known concentrations (0.039–5 μM) were introduced into the device. Cy5 intensity was measured with a microarray scanner (LS Reloaded, Tecan) using a 633-nm laser and a 695-nm filter, and a calibration curve was plotted. The concentration of DNA in each chamber was then calculated according to this standard curve.

Kedmi A., Zehavi Y., Glick Y., Orenstein Y., Ideses D., Wachtel C., Doniger T., Waldman Ben-Asher H., Muster N., Thompson J., Anderson S., Avrahami D., Yates JR I.I.I., Shamir R., Gerber D, & Juven-Gershon T. (2014). Drosophila TRF2 is a preferential core promoter regulator. Genes & Development, 28(19), 2163-2174.

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Quantifying CRISPR-Cas9 Cleavage Activity

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Biotinylated and Cy5-labeled dsDNA products were diluted to 50 nM with 10 nM of free biotinylated primer to compete with the matching dsDNA product during the surface binding process. This mixture was then introduced to the NeutrAvidin-coated surface below the MITOMI valve, together with the biotinylated primers. The gradient signal intensity was determined by the microarray fluorescent scanner (LS Reloaded, TECAN) before the exposure to CAS9. The concentration of each button was determined by a Cy5-primer calibration curve of 100, 50, 25 and 0 nM diluted in PBS, which was introduced to the side chambers and scanned for their fluorescent signal. The cleavage activity of CAS9 was determined using Proteinase K (Thermo-Scientific, Waltham, Massachusetts, USA, #EO0491, 1:40 in PBS) which was incubated with open MITOMI valves for 10 min at 42 °C. The difference in the signals before and after the incubation with Proteinase K and degradation of CAS9 were calculated, subtracting the background fluorescence level (which changed due to bleaching and Proteinase K degradation of the surface). The normalized calculated delta of signals was translated to nM using the calibration curve described above.

Peleg-Chen D., Shuvali G., Brio L., Ifrach A., Iancu O., Barbiro-Michaely E., Hendel A, & Gerber D. (2022). Microfluidic tool for rapid functional characterization of CRISPR complexes. New Biotechnology, 68, 1-8.

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Quantitative Protein-DNA Interaction Assay

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Protein-DNA interactions were carried out as previously described (Glick et al., 2016b (link)). In each experiment, 25 mL of extract (50 ng of protein) was loaded into the device. Introduction of 3′-HIS&5′-cMyc or Flag-tagged TFs complex into the DNA chambers solubilize spotted DNA, allowing TFs and DNA to interact. TF-DNA complexes were then captured on the chip surface beneath the ‘button’ valve during a 1 hour incubation period at 32°C. Next, MITOMI was performed by closing the ‘button’ valve to trap the interactions. Protein complexes and DNA not trapped by MITOMI were then washed away. TFs were labeled with α-FLAG cy3 (Sigma). Proteins expression levels and interacting DNA signals were measured with a microarray scanner (LS Reloaded, Tecan) using a 488 nm laser and 535 filter for Cy3, and a 633 nm laser and 695 nm filter for Cy5. Cy3 intensities under the ‘button’ valve reflect the number of surface-bound protein molecules; Cy5 intensities under the ‘button’ valve reflect the number of DNA molecules bound by surface-immobilized protein. The ratio of Cy5 to Cy3 fluorescence is proportional to the number of DNA molecules bound per protein, namely, protein fractional occupancy. Cy5 intensities within the DNA chamber reflect the amount of soluble DNA available for binding.

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On-Chip Protein Expression and Detection

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For on-chip expression, a pre-mixed reticulocyte lysate supporting protein expression by T7 promoter (12.5 μl) with or without microsomal membranes was flowed into the DNA chambers, following surface chemistry. See more details in refs. ^{8 (link),21 (link)}. Alternatively, in vitro translation took place in tube, and the lysate was flowed directly into the protein chambers for immobilization. Detection of the immobilized proteins was based on immunofluorescence with either Cy3-coupled anti-Myc antibodies (1/100 dilution; Sigma Israel) or Alexa-Fluor 647-coupled anti-His antibodies (Qiagen, Venlo, Netherlands). The detection antibodies were flowed into the device, and incubated with the immobilized proteins under the button for 30 min at RT, followed by a wash with PBS buffer. Protein expression levels were determined with a microarray scanner (LS Reloaded, Tecan, Männedorf, Switzarland) using a 532 nm laser and 575/50 nm filter for Cy3-anti-Myc, and 633 nm laser and 692/40 filter for Alexa 647-anti-His antibodies.

Nevenzal H., Noach-Hirsh M., Skornik-Bustan O., Brio L., Barbiro-Michaely E., Glick Y., Avrahami D., Lahmi R., Tzur A, & Gerber D. (2019). A high-throughput integrated microfluidics method enables tyrosine autophosphorylation discovery. Communications Biology, 2, 42.

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Quantifying On-Chip DNA Concentration

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To determine actual on-chip printed DNA concentration, Cy5 labeled oligonucleotides with known concentrations (0.005–1 μM) were introduced into the device. Cy5 intensity was measured with a microarray scanner (LS Reloaded, Tecan) using 633 nm laser and 695 nm filter and a calibration curve was plotted. The concentration of spotted DNA in each chamber was then calculated according to this standard curve.

Glick Y., Orenstein Y., Chen D., Avrahami D., Zor T., Shamir R, & Gerber D. (2015). Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities. Nucleic Acids Research, 44(6), e51.

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Microarray Image Analysis Protocol

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LS Reloaded microarray scanner (LS Reloaded; Tecan, Männedorf, Switzarland) and GenePix7.0 (Molecular Devices) image analysis software were used for analysis and presentation of the images for the figures.
For more details, see Noach-Hirsh et al. (2015) (link).

Wasserman D., Nachum S., Cohen M., Enrico T.P., Noach-Hirsh M., Parasol J., Zomer-Polak S., Auerbach N., Sheinberger-Chorni E., Nevenzal H., Levi-Dadon N., Wang X., Lahmi R., Michaely E., Gerber D., Emanuele M.J, & Tzur A. (2020). Cell cycle oscillators underlying orderly proteolysis of E2F8. Molecular Biology of the Cell, 31(8), 725-740.

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Fluorescent Measurement Using Microarray

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We performed fluorescent measurements using microarray scanner (LS Reloaded, Tecan, sensitivity of less than 0.1 Fluorophore equivalent/µm²). Alexa Fluor 467 was excited with a laser at 632 nm, and emission signals were collected through a filter at 692 nm ± 25 nm.

Wissberg S., Ronen M., Oren Z., Gerber D, & Kalisky B. (2020). Sensitive Readout for Microfluidic High-Throughput Applications using Scanning SQUID Microscopy. Scientific Reports, 10, 1573.

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