Yeast expressing the indicated AD and BD constructs was grown overnight at 30°C in 5 mL YPD and then diluted to 0.2 OD600 in YPD for 90 minutes. Whole-cell lysates of equal cell numbers (0.2 OD600) were extracted by TCA precipitation. Protein was separated on 10% acrylamide gels and transferred to PVDF membranes, and protein was detected on a LiCor CLX scanner. RAD51C expression was detected with RAD51C antibody (ab55728 1:500; Abcam), equal loading was detected using a Kar2 antibody (sc-33630 1:2,000; Santa Cruz Biotechnology) and IR dye secondary antibodies (1:20,000) from LiCor Biosciences. The image was adjusted for brightness and contrast using Photoshop (Adobe Systems Inc.).
Clx scanner
Manufactured by LI COR
Sourced in United States
The CLx scanner is a laboratory equipment designed for high-throughput imaging. It utilizes fluorescence detection technology to capture and analyze images of biological samples. The core function of the CLx scanner is to provide researchers with a reliable and efficient tool for visualizing and quantifying various molecular targets within their samples.
Automatically generated - may contain errors
Lab products found in correlation
Irdye secondary antibody, by LI COR (2 mentions)
Ab55728, by Abcam (2 mentions)
Forty plex human cytokine microarray, by RayBiotech (2 mentions)
Protran, by Cytiva (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Prism 7, by GraphPad (1 mentions)
Prefer fixative, by Anatech (1 mentions)
Image studio v2, by LI COR (1 mentions)
Syto60, by Thermo Fisher Scientific (1 mentions)
Image studio lite software, by LI COR (1 mentions)
α tubulin, by Merck Group (1 mentions)
γ tubulin, by Merck Group (1 mentions)
Nucleolin, by Abcam (1 mentions)
Apyrase, by Merck Group (1 mentions)
Image studio software package, by LI COR (1 mentions)
11 protocols using clx scanner
1
Western Blot Analysis of RAD51C
2
Western Blot and RPPA Analysis
3
GPCR Activation Assay in HEK293 Cells
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HEK293 cells stably expressing HA-GIPR-EFGP or HA–GLP-1R–EFGP clones were plated into poly-D-lysine–coated 96-well microplates and cultured until cells reached 80%–90% confluency. On the day of assay, growth media was removed, and cells were rinsed once with prewarmed starvation media (growth media without serum or antibiotics, supplemented with 0.1% casein) and equilibrated with fresh media for 1 hour at 37°C, 5% CO2. Concentration response curves of GLP-1, GIP, and tirzepatide were prepared in prewarmed starvation media, added to cells for designated times, and incubated at 37°C. At the end of the study, media was removed, and cells were placed on ice and fixed with Prefer fixative (Anatech) for 10 minutes. Fixative was removed, and cells were washed in PBS and blocked with Odyssey blocking buffer (Licor) for 1 hour. Cells were incubated with anti-HA/DyLight800 antibody (1:700) (Rockland Immunochemicals, 600-445-384) for 1 hour followed by washes with PBS-T. Plates were scanned using a Licor Clx scanner with the 800 nm channel laser to capture fluorescence signal in each well. Data were normalized to maximum concentrations of GLP-1 or GIP (100%) and no ligand (0%) and analyzed by nonlinear regression (sigmoidal concentration-response) and plotted using GraphPad Prism 7 software.
4
Forty-plex Cytokine Microarray Analysis
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Forty-plex human cytokine microarray (RayBiotech, catalog #: QAH-CYT-4) was employed to further test the efficacy of plasmonic-fluor. To begin with, the glass substrate of the microarray was blocked with 100 μl sample diluent (catalog #: QA-SDB) followed by incubation with sample standard (catalog #: QAH-CYT-4-STD) at room temperature for 2 hours with gentle rocking. The microarray was washed by five times using 1X wash buffer I (catalog #: AA-WB1-30ML) followed by twice washing with 1X wash buffer II (catalog #: AA-WB2-30ML). Next, 80 μl of reconstituted detection antibody cocktail was added into each well and incubated for another 2 hours under gentle rocking. Following the incubation, washing process was repeated again as described above. Subsequently, 80 μl of 800CW-streptavidin (50 ng/ml in 1% BSA) was added to the array slide and incubated for 20 minutes, washed, and immersed with plasmonic-fluor-800CW (extinction~1) for one hour. The slide was scanned using LI-COR CLx scanner with the following parameters: laser power~3.5; resolution~21 μm; channel: 800; height: 1.8 mm.
5
Transwell Assay for Monocyte Migration
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THP1 monocytes were differentiated in 100 ng/mL TPA complete medium for 48 h and allowed to recover for 24 h on transwell inserts. Respective siRNA knockdowns were performed on iCECS (iCECANGPTL4, iCECTTP and iCECCtrl). Inserts containing differentiated THP1 cells were then introduced to the transfected iCECs and exposed to various pro- and anti- inflammatory stimuli for 10 h (Fig. 3e ). Inserts were then washed with PBS twice and fixed in 1% glutaraldehyde for 10 min, rinsed with PBS and stained with SYTO 60 (Thermo Fisher, USA) for 30 min. Cotton buds were used to remove all unmigrated cells trapped in the upper chamber of the inserts. Inserts were rinsed again in PBS before the quantification of fluorescence using the CLx scanner and Image Studio V2.1 (LI-COR Biosciences, USA). Relative fluorescence as a readout for cellular migration was calculated by normalizing the intensities between test wells (THP1 and iCECs) and control wells (THP1 without iCECs).
6
Forty-plex Cytokine Microarray Analysis
7
Chaperone Expression Quantification
8
Quantitative Western Blotting of Embryonic Proteins
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Protein samples consisting of five fish/sample were collected at appropriate stages. Embryos were homogenized and suspended in sample buffer containing Tris-HCl pH 8.0, NaCl, SDS, sodium deoxycholate, NP-40 and protease inhibitor and used for western blotting according to standard protocols (Dash et al., 2021 (link)). Protein quantity was estimated via a bicinchoninic acid (BCA) assay. The primary antibodies used were: γ-tubulin (1:1000, Sigma-Aldrich, T6557), α-tubulin (1:10,000, Sigma-Aldrich, T5168), p53 (1:500, Cell Signaling Technology, 2524S) and Nucleolin (1:500, Abcam, ab22758). Western blots were imaged and quantified using a CLx-Scanner (Li-COR) and Odyssey Software. For quantification, band intensities for p53 were compared with the housekeeping control protein γ-tubulin. Two-tailed, paired Student's t-test was performed for statistical analysis.
9
Ubiquitin Discharge Assay Protocol
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E2 enzymes were charged with ubiquitin under the following conditions: 1 μM E1, 1 μM UbiquitinAtto, 3 mM ATP, 4 μM E2 (reaction buffer: 50 mM Hepes, pH 7.5, 150 mM NaCl, 20 mM MgCl2, and 5 mM CaCl2). The reactions were incubated at 25°C for 20 min, followed by a 5-min incubation with 1U of apyrase (Sigma-Aldrich). Discharge was initiated by addition of L-lysine, at a final concentration of 20 mM, and E3, at a final concentration of 4 μM. Discharge assays were incubated at 25°C for up to 60 min, the samples were quenched by addition of SDS sample buffer and flash-freezing on dry ice at the described time intervals, and resolved by SDS–PAGE. For quantification, the gels were scanned with a LICOR CLx scanner, and the bands for E2–UbAtto were integrated using the ImageStudio software package (LI-COR). Experiments were performed in duplicate (UBE2E2) or triplicate (UBE2D and UBE2E1). The scans were converted to greyscale.
10
Quantifying Nuclear RAD51C Levels
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