G box chemi xr5

Manufactured by Syngene

Sourced in United Kingdom, United States

The G:BOX Chemi XR5 is a compact, high-performance imaging system designed for capturing and analyzing chemiluminescent, fluorescent, and visible light signals. It features a large sample area, high-resolution camera, and advanced software for image acquisition and analysis.

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Lab products found in correlation

A5441, by Merck Group (4 mentions) Enhanced chemiluminescence horseradish peroxidase substrate detection kit, by Merck Group (3 mentions) Enhanced chemi luminescence (ecl), by Bio-Rad (3 mentions) Clarity western ecl, by Bio-Rad (3 mentions) Pvdf membrane, by Merck Group (3 mentions) Genetools image analysis software, by Syngene (2 mentions) Image studio lite, by LI COR (2 mentions) Pab204, by Merck Group (2 mentions) β actin, by Merck Group (2 mentions) Bradford protein assay, by Thermo Fisher Scientific (1 mentions) Ponceau s, by Merck Group (1 mentions) Glass bead, by Biospec (1 mentions) Mini beadbeater 16, by Biospec (1 mentions) Ha 11, by BioLegend (1 mentions) Odyssey clx imager, by LI COR (1 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Ab105381, by Abcam (1 mentions) Protease inhibitors cocktail, by Merck Group (1 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions) M20010, by Abmart (1 mentions)

35 protocols using g box chemi xr5

Immunoblotting Analysis of PD-1 and PD-L1

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Fifty milligrams of proteins were resolved on SDS electrophoresis gels with the use of standard procedures [37 (link)]. Immunoblotting was performed with anti-PD-1 rabbit antibody, PRS4065 (Merck, Darmstadt, Germany) or with anti-PD-L1 rabbit antibody, PRS4059 (Merck, Darmstadt, Germany). Final detection was achieved with the appropriate secondary antibody coupled to horseradish peroxidase (HRP), A0545 and anti-rabbit IgG-HRP (Merck, Darmstadt, Germany), followed by luminol-based chemiluminescent substrate (Cyanagen, Bologna, Italy) and visualized by a gel imaging system for chemiluminescence G: Box Chemi XR5 (Syngene, Cambridge, UK).
Equal loading of the proteins was additionally checked by the immunodetection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody, MAB 374 (Merck Millipore, Burlington, MA, USA) and anti-mouse IgG-HRP, with A4416 as a secondary antibody (Sigma-Aldrich, Saint Louis, MO, USA), and visualized by G: Box Chemi XR5 (Syngene, Cambridge, UK). For the analysis of the protein band density, ImageJ 1.53k software (Wayne Rasband, USA) was used.

Malinowska K., Kowalski A., Merecz-Sadowska A., Paprocka-Zjawiona M., Sitarek P., Kowalczyk T, & Zielińska-Bliźniewska H. (2023). PD-1 and PD-L1 Expression Levels as a Potential Biomarker of Chronic Rhinosinusitis and Head and Neck Cancers. Journal of Clinical Medicine, 12(5), 2033.

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Muscle Protein Quantification and Western Blotting

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Briefly, muscle were homogenised in 10-fold volume excess of ice-cold sucrose lysis buffer (Additional file 1: Table S3). Protein concentration was determined using the Bradford protein assay (ThermoScientific). Forty microgrammes of protein was loaded into 4–12% Bis-Tris midi protein gels (Invitrogen) prior to electrophoresis. Proteins were transferred and blocked in blocking buffer (Additional file 1: Table S3) before incubation with primary antibodies (Additional file 1: Table S4) overnight at 4 °C. Membranes were then incubated in horseradish peroxidase-conjugated secondary antibody (1/10,000) at room temperature for 1 h. Antibody detection was performed via enhanced chemiluminescence horseradish peroxidase substrate detection kit (Millipore). Imaging was undertaken using a G:Box Chemi-XR5 (Syngene) and band quantification via (ImageJ). All data were corrected for protein loading as determined after Ponceau S staining (Sigma-Aldrich).

Fenton C.G., Webster J.M., Martin C.S., Fareed S., Wehmeyer C., Mackie H., Jones R., Seabright A.P., Lewis J.W., Lai Y.C., Goodyear C.S., Jones S.W., Cooper M.S., Lavery G.G., Langen R., Raza K, & Hardy R.S. (2019). Therapeutic glucocorticoids prevent bone loss but drive muscle wasting when administered in chronic polyarthritis. Arthritis Research & Therapy, 21, 182.

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Steady-State Protein Level Quantification

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To assess steady-state protein levels, 2 OD₆₀₀ equivalent of mid-log cells was harvested by low-speed centrifugation and the pellets flash-frozen in liquid nitrogen. Cell pellets were thawed on ice and resuspended in 200 µL of 20 mM NaN₃ with 1 mM phenylmethylsulfonyl fluoride. Glass beads (BioSpec, Bartlesville, OK) and 200 µL 5× SDS-PAGE sample buffer were added before lysis at 4°C using a Mini-Beadbeater-16 (BioSpec). Samples were heated for 5 min at 95°C, cleared by centrifugation for 3 min at 14,000 × g, and resolved by SDS-PAGE. Immunoblots were developed with Supersignal Pico chemiluminescent substrate (Pierce Chemical, Rockford, IL), imaged using a G:BOX Chemi XR5 (Syngene, Frederick, MD), and quantified with GeneTools image analysis software (Syngene). Statistical analyses were performed with Prism (Graphpad, La Jolla, CA) or Excel (Microsoft, Redmond, Wa).

Margulis N.G., Wilson J.D., Bentivoglio C.M., Dhungel N., Gitler A.D, & Barlowe C. (2016). Analysis of COPII vesicles indicates a role for the Emp47-Ssp120 complex in transport of cell surface glycoproteins. Traffic (Copenhagen, Denmark), 17(3), 191-210.

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Antibody Detection and Quantification

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For primary antibodies, rabbit polyclonal antiserum against Erv41, Erv46, and Och1 (Otte et al., 2001 ); Yet3 (Wilson and Barlowe, 2010 (link)); Fpr2; and Gls1 (Shibuya et al., 2015 (link)) has been described previously. Mouse monoclonal antibody HA.11 against the hemagglutinin epitope was purchased from BioLegend (San Diego, CA) and used for co-IPs and detection of HA-tagged proteins by immunoblot. Primary antibodies were used at 1:1000 dilutions except for anti-Fpr2 and anti-HA, which were used at 1:500 and 1:2000 dilutions, respectively. For secondary antibodies, two different systems were used. First, horseradish peroxidase–linked anti-rabbit and anti-mouse antibodies (GE Healthcare) were used at 1:10,000 dilutions. Blots were developed with SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific), imaged using a G:Box Chemi XR5 (Syngene), and quantified with GeneTools image analysis software (Syngene). Alternatively, IRDye cross-linked anti-rabbit and anti-mouse antibodies (LI-COR Biosciences) were used at 1:10,000 dilutions. Blots were imaged with an Odyssey CLx imager (LI-COR Biosciences) and quantified with Image Studio Lite (LI-COR Biosciences).

Keiser K.J, & Barlowe C. (2020). Molecular dissection of the Erv41-Erv46 retrograde receptor reveals a conserved cysteine-rich region in Erv46 required for retrieval activity. Molecular Biology of the Cell, 31(3), 209-220.

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Western Blot Protein Detection Protocol

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Western blot was performed as previously described [42 (link)]. Briefly, cells were lysed in the radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% (v/v) NP-40, 0.5% (w/v) Sodium deoxycholate, 0.1% (w/v) SDS] with protease inhibitors cocktail (Sigma) added freshly. The lysates were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore), which were blocked in 5% milk for 1 hour and then probed with antibody against TCTN1(1:200; ab105381; Abcam), or actin (1:4000; M20010; Abmart) as a loading control. Blots were developed with Immobilon Western Chemiluminescent HRP Substrate (Millipore) and visualized on G: Box Chemi XR5 (Syngene).

Meng D., Chen Y., Zhao Y., Wang J., Yun D., Yang S., Chen J., Chen H, & Lu D. (2014). Expression and prognostic significance of TCTN1 in human glioblastoma. Journal of Translational Medicine, 12, 288.

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Quantifying Lysosomal Enzyme Activity

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Fibroblasts were collected and lysed, and protein concentration determined. For labelling, equal amounts of protein were incubated with cy5-labeled ABP ME569 in 150 mM McIlvaine buffer, pH 5.2 for 1 h at 37 °C. Samples were denatured with 4× Laemmli buffer, boiled for 5 min at 98 °C and resolved by SDS-PAGE using the TGX gel system (Bio-Rad). The gels were placed in an imager (G:BOX ChemiXR5, Syngene) and fluorescence was detected with red LED lightning modules/705 M filter. The amount of labeled GCase was quantified using GeneTools v.4.03.01.0 (Syngene).
For visualization, fixed cells were incubated with ABP MDW933 (green) and images were acquired using the AXIO (Zeiss) equipped with AxioCam MRm (LED ex 470 nm, em 500-550 nm). Images were analyzed using freeware program Fiji (an image processing package to ImageJ, https://fiji.sc/).

Fog C.K., Zago P., Malini E., Solanko L.M., Peruzzo P., Bornaes C., Magnoni R., Mehmedbasic A., Petersen N.H., Bembi B., Aerts J.F., Dardis A, & Kirkegaard T. (2018). The heat shock protein amplifier arimoclomol improves refolding, maturation and lysosomal activity of glucocerebrosidase. EBioMedicine, 38, 142-153.

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Ubiquitin Pull-Down and Western Blotting

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Cell lysates were prepared in 1x NuPAGE LDS sample buffer containing 2-mercaptoethanol (final concentration 1.5%) and left to denature overnight at room temperature. Ubiquitin pull-down samples and prepared cell lysates (25-75 μg of total protein) were loaded into 4-12% Bis/Tris precast gels (ThermoFisher Scientific, Leicestershire, UK) prior to SDS-PAGE. Gels were run in 1x MOPS buffer for approximately 80 minutes at 150V. Proteins were transferred onto PVDF membranes (Millipore, Hertfordshire, UK) for 1h at 100V. Membranes were blocked in 3% BSA diluted in Tris-buffered saline Tween-20 (TBS-T):137 mM sodium chloride, 20 mM Tris-base 7.5 pH, 0.1% Tween-20 for 1 h and incubated overnight at 4°C with the appropriate primary antibody. Primary antibodies were diluted in 3% BSA made up in TBS-T (see Supplementary Table 1). Membranes were washed in TBS-T three times prior to incubation in horse radish peroxidase-conjugated secondary antibodies (see Supplementary Table 1) at room temperature for 1h. Membranes were washed a further three times in TBS-T prior to antibody detection using enhanced chemiluminescence horseradish peroxidase substrate detection kit (Millipore, Hertfordshire, UK). Imaging was undertaken using a G:Box Chemi-XR5 (Syngene, Cambridgeshire, UK). Quantification was performed using ImageJ/Fiji (NIH, Bethesda, MD, USA).

Seabright A.P., Fine N.H., Barlow J.P., Lord S.O., Musa I., Gray A., Bryant J.A., Banzhaf M., Lavery G.G., Hardie D.G., Hodson D.J., Philp A, & Lai Y. (2020). AMPK activation induces mitophagy and promotes mitochondrial fission while activating TBK1 in a PINK1‐Parkin independent manner. The FASEB Journal, 34(5), 6284-6301.

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SDS-PAGE and Western Blotting Protocol

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Equivalent amounts of protein (30 ug) were separated on 7.5–12.5% gels by SDS-page as previously described [26] (link), proteins transferred to BioTrace NT nitro-cellulose membranes (Pall Life Sciences, Pensacola, FL, USA), blocked in 3% milk/TBST and incubated overnight in primary antibodies and subsequently incubated for 1 h at room temperature in relevant secondary antibodies. Chemiluminescence horseradish peroxidase reagent kit (Merck-Millipore) was used to quantify protein content following IgG binding. Images were captured with a G: Box Chemi-XR5 (Syngene (A Division of Synoptics Ltd.), Cambridge, UK) imaging system while blot bands were quantified using GeneTools software (Syngene).

Dent J.R., Martins V.F., Svensson K., LaBarge S.A., Schlenk N.C., Esparza M.C., Buckner E.H., Meyer G.A., Hamilton D.L., Schenk S, & Philp A. (2017). Muscle-specific knockout of general control of amino acid synthesis 5 (GCN5) does not enhance basal or endurance exercise-induced mitochondrial adaptation. Molecular Metabolism, 6(12), 1574-1584.

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Cell Signaling in cECM-Silk Scaffold Assimilation

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In order to assess the potential cell signaling pathways involved in cECM incorporation into silk scaffolds, pathways previously implicated in proliferation and cell survival were evaluated by Western blot techniques previously described by our lab.^{32 (link)} Equal amounts of protein (12 μg per lane) were loaded into pre-cast 4–12% polyacrylamide gels (456-1086) and run before being transferred to a nitrocellulose membrane. Primary antibodies were used to evaluate the expression of the various proteins over time (day 2 and day 6) and for each condition. β-actin was utilized to normalize to total cell content. Antibodies were used at a 1/1000 dilution for primary antibodies and a 1/5000 dilution for secondary antibodies. Species-specific secondary antibodies conjugated to HRP (cat #s 656120 and 656520, Invitrogen, Carlsbad, CA) were used for enhanced chemiluminescence. Images were acquired on the G:Box Chemi XR5 (Syngene, Cambridge, United Kingdom). Expression intensities were analyzed using ImageJ (NIH, Bethesda, MD). Expression intensities for each protein were normalized to the mean expression intensity per blot for that respective protein to allow for comparison between blots.

Stoppel W.L., Gao A.E., Greaney A.M., Partlow B.P., Bretherton R.C., Kaplan D.L, & Black LD I.I.I. (2016). Elastic, silk-cardiac extracellular matrix hydrogels exhibit time-dependent stiffening that modulates cardiac fibroblast response. Journal of biomedical materials research. Part A, 104(12), 3058-3072.

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Western Blot Analysis of Cre Induction

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Western blot analysis was performed on fully-confluent Cre-responsive cell lines harvested from 24-well plates 6 or 22 hours after the single administration of PTD-Cre (15 nM) or syn-mRNA-Cre (2.1 nM). The cells were lysed using RIPA buffer composed of 150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0 [27 (link)]. Cell lysates (14 μg total protein per well) were mixed with 4x Laemmli loading buffer containing 8% SDS, 40% glycerol, 0.02% bromophenol blue, 250 mM Tris, and 20% 2-mercaptoethanol (all from Sigma-Aldrich), pH 6.8, heated at 95°C for 3 min, and run on a 15% polyacrylamide gel and transferred to PVDF membranes (Merck Millipore) using a Pierce G2 electroblotter (Thermo Fisher Scientific). The membranes were blocked with 3% BSA (Sigma-Aldrich). Primary antibodies included a rabbit anti-T7 antibody (Abcam, Cambridge, UK) for detecting Cre recombinase (1:2000 dilution) and a mouse anti-beta-actin antibody (Sigma-Aldrich) as a loading control (1:7500 dilution). The secondary antibodies included goat anti-rabbit IgG-HRP (Merck Millipore) and rabbit anti-Mouse IgG-HRP (Thermo Fisher Scientific), each diluted 1:50000. Chemiluminescent SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific) was used for detection. The signals were acquired using a G:BOX Chemi XR5 (Syngene, Cambridge, UK).

Leontovyc I., Habart D., Loukotova S., Kosinova L., Kriz J., Saudek F, & Koblas T. (2017). Synthetic mRNA is a more reliable tool for the delivery of DNA-targeting proteins into the cell nucleus than fusion with a protein transduction domain. PLoS ONE, 12(8), e0182497.

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