Infiltrated leaves expressing GFP or GB1-GFP were harvested at 3, 5, and 7 DPI. The GFP fluorescence image of leaves was captured using the LAS3000 system (Fujifilm, Tokyo, Japan). The density of GFP fluorescence from the whole infiltrated leaves was measured by the official software of LAS3000 system (Fujifilm, Tokyo, Japan).
Las 3000 system
Manufactured by Fujifilm
Sourced in Japan, United Kingdom
The LAS-3000 system is a compact, high-performance imaging system designed for capturing and analyzing images of various biological samples, such as gels, blots, and membranes. The core function of the LAS-3000 system is to provide users with a versatile and efficient tool for imaging and quantifying their experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (6 mentions)
Anti β actin, by Merck Group (4 mentions)
Complete protease inhibitor cocktail, by Roche (4 mentions)
β actin, by Merck Group (4 mentions)
Nitrocellulose membrane, by Cytiva (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Peroxidase labeled secondary antibody, by Agilent Technologies (3 mentions)
Exoab cd63a 1, by System Biosciences (3 mentions)
Image reader las 3000, by Fujifilm (2 mentions)
Supersignal west pico ecl, by Thermo Fisher Scientific (2 mentions)
Clarity western ecl, by Bio-Rad (2 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (2 mentions)
Lumi light western blotting substrate, by Roche (2 mentions)
Anti flag, by Merck Group (2 mentions)
Flotillin 1, by BD (2 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
β actin, by Enogene (2 mentions)
74 protocols using las 3000 system
1
Quantifying GFP Expression in Leaves
2
Western Blot Analysis of Protein Signaling
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Protein concentrations were determined according to the Bradford method using BSA as standard. Cell lysates (10–20 μg per sample) were then separated by 5–20% SDS-PAGE with known molecular weight markers (Bio-Rad, California, USA) and transferred onto polyvinylidene fluoride membranes by standard procedures. Membranes were incubated in blocking solution (5% skim milk or BSA in TBS-T) for 3 h at room temperature, and hybridized overnight at 4°C with primary antibodies against vinculin (Sigma) (1:4000), AdipoR1 (Immuno-Biological Laboratories Inc., Minneapolis MN) (2 μg /ml), and also Akt (1:1000), phospho-Akt (s473 or t308) (1:1000), AMPK (1:1000), phospho-AMPK (1:1000) all from Cell Signaling Technology (CST, Danvers, MA, USA). The membranes were washed three times with TBS-T and then incubated with either anti-rabbit (Sigma) (1:10000) or anti-mouse (Abcam, Cambridge, U.K.) (1:10000) secondary antibodies for 1 h at room temperature. After washing, immunodetection was performed using an enhanced chemiluminescence solution (Clarity Max Western ECL Substrates, Bio-Rad) and scanned on Fujifilm LAS-3000 system and quantified using Image-J software (National Institutes of Health).
3
Microglia Protein Expression Analysis by Western Blot
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Denaturing SDS-PAGE (BioRad) gels were used to separate 8 µg of microglia protein per lane. Proteins were transferred to nitrocellulose, which was subsequently blocked with 5% milk Tris-buffered saline tween-20. Blots were probed for Sema4A, Hft, or beta-actin for 16 hr at 4℃. Anti-Sema4A (1:200, rabbit polyclonal antibody) was purchased from Abcam. Anti-Hft (1:1,000, rabbit monoclonal antibody) was purchased from Cell Signaling (Danvers, MA). Anti-beta-actin (1:3,000, mouse monoclonal antibody) was purchased from Sigma. Corresponding anti-rabbit or anti-mouse HRP-conjugated secondary antibodies were used (1:5,000, GE Amersham, Amersham, UK). Protein was visualized with ECL reagents (Perkin–Elmer, Waltham, MA) on the FujiFilm LAS-3000 System and Image Reader LAS-3000 software. Densitometry quantification was performed using MultiGauge software.
4
Analysis of EGFR-mediated Signaling Pathways
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After serum-starving for 24 hours, H1299-EGFR-WT and H1299-EGFR-L858R cells were treated with 40 ng/ml EGF for 5 minutes, with or without pretreatment with the pharmacological inhibitors, AG1478 (EGFR inhibitor), AG490 (JAK-STAT3 inhibitor), LY294002 (PI3K-AKT inhibitor), U0126 (ERK inhibitor) or vehicle control (DMSO) for 1 hour. Cells lysates were prepared and analyzed by Western blotting as described previously50 (link). The following primary antibodies were used for Western blot analyses: anti-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-Y992-EGFR (Cell Signaling Technology, Beverly, MA), anti-phospho-Y705-STAT3 (Cell Signaling Technology), anti-STAT3 (Cell Signaling Technology), anti-phospho-S473-AKT (Cell Signaling Technology), anti-AKT (Cell Signaling Technology), anti-ERK1/2 (Invitrogen), and anti-phospho-T202/Y204-ERK (BD Biosciences, San Diego, CA). Recombinant human EGF, CXCL12 (SDF-1α), CXCR4 neutralizing antibody (12G5), monoclonal anti-CXCR4 antibody, and mouse IgG2B isotype control antibody were purchased from R&D Systems (Minneapolis, NM). Chemiluminescent signals on western blots were captured using the Fujifilm LAS 3000 system (Fujifilm) and image processing was performed with Adobe Photoshop software (Adobe Systems).
5
Western Blot Analysis of Cell Lysates
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Cultured HEPG2, H1581, and THP-1 cells were lysed in Triton lysis buffer (14 (link)) or radioimmunoprecipitation buffer with cOmplete Protease Inhibitor Cocktail (catalog no.: 11697498001; Roche). SDS-PAGE samples were prepared with lysates mixed with Laemmli buffer containing β-mercaptoethanol, followed by boiling at 95 °C for 5 min. Western blot images were acquired with SuperSignal West pico ECL (catalog no.: 34580; Thermo Fisher Scientific) or Clarity Western ECL (catalog no.: 1705061; Bio-Rad) using LAS-3000 system (FujiFilm).
6
Western Blot Protein Quantification Protocol
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For Western blot analysis, 5 × 106 cells were harvested and proteins were solubilized as described.48 (link) 30 µg protein/lane was separated in 10% SDS-PAGE gels, transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) and stained with Ponceau S as previously described.45 (link) Membranes were incubated overnight at 4°C with a primary polyclonal antibodies directed against BGN (Proteintech, Rosemont, IL, USA) and K-RAS (Cell Signaling Technology, Danvers, USA) and monoclonal antibodies directed against β-actin (Sigma-Aldrich, St. Louis, USA) and/or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology), respectively, followed by incubation for 1 h with a horseradish peroxidase (HRP) linked secondary antibody (Ab, Cell Signaling Technology) and developed using the ECL method. Chemiluminescence signals were visualized with the Lumi-Light Western Blotting Substrate (Roche Diagnostics) and recorded with a LAS3000 system (Fuji, Tokyo, Japan). To quantify the protein expression, the appropriate area of the signal was integrated using an AIDA image analyzer (Raytest, Straubenhardt, Germany) and subsequently normalized to β-actin or GAPDH.
7
Protein Extraction and Western Blot Analysis
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The cells were lysed by 1x Laemmli buffer containing protease inhibitors (0.5mM PMSF and 1x Complete Protease Inhibitor Cocktail; Roche) and phosphatase inhibitors (50mM NaF, 1.5mM Na3VO3). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) before transferring to nitrocellulose membranes. SDS-PAGE was performed according to published protocol [21 ]. The following antibodies were used: anti-Flag (Sigma, 1:2000), anti-HA antibody (ProteinTech, 1:1000) and anti-beta actin (Sigma, 1:5000). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Pierce. Chemiluminescent reagents (Pierce) were used to visualize the protein signals under the LAS-3000 system (Fujifilm).
8
Mosquito Salivary Gland and Midgut Protein Analysis
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The salivary gland and midgut of each female mosquito sample were dissected. The detailed procedures are described in detail [15 (link)]. Total proteins (40 µg) were extracted from the salivary gland and midgut using Tissue protein extraction reagent (Thermo Scientific, Wilmington, Delaware, USA). We loaded the proteins in a 10% acrylamide gel with sample buffer. The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and then blocked the membrane with 5% milk TBS/T solution for 1 hr room temperature. Anti-heparin was used as the primary antibody. Monoclonal anti-β-actin (Santi Cruz Technology, Santa Cruz, California, USA) was used for loading control. Bound antibody was detected by using the Supersignal West Pico Chemiluminescent Substrate (Pierce, Rockford, Illinois, USA). Chemiluminescent signals were captured with the LAS 3000 system (Fuji, Tokyo, Japan).
9
Western Blot Analysis of MCPH1 Protein
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To determine the effect at protein level, control and gene‐edited cells were lysed in NETN‐300 buffer containing protease inhibitor tablets (Complete Mini EDTA‐free, Roche Diagnostics) and phosphatase inhibitor cocktail (Set V, Merck). Freshly prepared cell lysates were denatured in Laemmli buffer and separated by SDS‐PAGE using 4–15% Mini‐PROTEAN TGX gels (Bio‐Rad). Proteins were transferred onto Immobilon‐P PVDF membrane (Millipore) and the membranes were blocked with buffer containing 1% BSA and 1% milk powder. Primary MCPH1 antibody (R&D systems, AF3998) was a goat polyclonal antibody raised against the amino acids 1–250 mapping to the N‐terminus of MCPH1 of human origin. Anti‐β‐Actin antibody AC15 (Abcam) was used as a loading control. Secondary antibodies were HRP‐conjugated AffiniPure Rabbit anti‐Goat and Goat anti‐Mouse IgG (H + L) antibodies (Jackson ImmunoResearch). The signal was detected using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) and Fujifilm LAS‐3000 system.
10
Western Blot Analysis of 5-LO, Akt, and IKK
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The levels of 5-LO expression, Akt and IKK phosphorylation were measured by Western blotting. Monocyte cell lysates were separated on 8% sodium dodecyl sulphate (SDS)-polyacrylamide gels, and transferred electrophoretically onto nitrocellulose membranes. Membranes were blocked with 5% skim milk in tris-buffered saline containing Tween 20 (TBST), and then incubated with anti-5-LO (1 : 1,000) in a blocking buffer. After the blots were incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody (1 : 3,000), chemiluminescence intensities were measured using the LAS-3000 SYSTEM (Fuji Photo Film, Japan). Membranes were re-blotted with an anti-β-actin antibody (MP Biomedicals, Aurora, Ohio) as an internal control.
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