The whole-cell lysate of BMMCs was subjected to SDS-PAGE, and the separated proteins were transferred to a membrane. The blots were then incubated with the target antibody, followed by the appropriate HRP-conjugated secondary antibody, and visualized by the LAS-3000 detection system (Fuji Photo Film, Japan). The following antibodies were used for immunoblot analyses: anti-Stat1 (Cell Signaling Technology; hereafter CST, #9172), anti-p-Stat1 (CST #7649), anti-Stat3 (CST #12640), anti-p-Stat3 (CST #9145), anti-PLCγ2 (Santa Cruz Biotechnology; hereafter SCB, sc-407), anti-p-PLCγ2 (CST #3871), anti-c-kit (SCB, sc-1494), anti-p-c-kit (CST #3319), anti-Syk (SCB, sc-929), anti-p-Syk (CST #2710), anti-Src (CST #2109), anti-p-Src family (CST #6943), anti-Phosphotyrosine (4G10)-HRP (MM 16–316), anti-FcεRI β chain (MM MABF38), anti-FcεRI γ chain (MM 06–727), anti-ERK1/2 (CST #4695), anti-p-ERK1/2 (CST #4370), anti-p38(CST #9212), anti-p-p38 (CST #9211), anti-β-Actin (CST #4970), and anti-α-Tubulin (CST #2125). To quantify the band intensities, Image Gauge (Fuji Photo Film, Tokyo, Japan) was used.
Las 3000 detection system
Manufactured by Fujifilm
Sourced in Japan, United States
The LAS-3000 is a detection system designed for laboratory use. It is capable of capturing and analyzing images from various sample types. The core function of the LAS-3000 is to provide high-quality image detection and analysis capabilities for scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Anti stat1, by Cell Signaling Technology (1 mentions)
Anti plcγ2, by Santa Cruz Biotechnology (1 mentions)
Image gauge, by Fujifilm (1 mentions)
Anti gst antibody, by Santa Cruz Biotechnology (1 mentions)
Sphingo strips, by Echelon Biosciences (1 mentions)
Supersignal west pico, by Fujifilm (1 mentions)
Hrp conjugated anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Enhanced chemiluminescent detection system, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Cytiva (1 mentions)
Anti p egfr, by Cell Signaling Technology (1 mentions)
Anti p met, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail set 5, by Merck Group (1 mentions)
Anti egfr, by Cell Signaling Technology (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail set 3, by Merck Group (1 mentions)
Cyclin a, by Santa Cruz Biotechnology (1 mentions)
Col1a1, by Santa Cruz Biotechnology (1 mentions)
7 protocols using las 3000 detection system
1
Western Blot Analysis of Signaling Proteins
2
Quantifying tPA Activity via Zymography
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tPA activity was measured using plasminogen-casein zymography. The culture supernatants were mixed with zymography buffer [200 mM Tris-HCl (pH 6.8), 8% w/v sodium dodecyl sulfate (SDS), 40% glycerol, 0.02% bromophenol blue, and without β-mercaptoethanol] and separated by electrophoresis on 8% polyacrylamide gels containing casein and plasminogen. After the electrophoresis, the gels were washed twice with 2.5% Triton X-100 for 30 min to eliminate the SDS. The gels were then incubated for 12–24 h at 37℃ in reaction buffer [20 mM Tris-HCl (pH 7.6) and 166 mM CaCl2,] for caseinolysis. To reveal the caseinolytic activity, the gels were stained with 0.1% Coomassie brilliant blue R-250 and destained using destaining solution (20% methanol, 10% acetic acid, and 70% deuterium-depleted water). tPA activity was visualized as the light bands that resulted from casein degradation. The caseinolysis band detected at 68 kDa matched the band of purified tPA standard that was present in the same gel. The gel was analyzed using a LAS 3000 detection system (Fujifilm, Minato-ku, Tokyo, Japan), and the band intensities were measured using ImageJ (https://imagej.nih.gov/ij/ ).
3
Lipid Binding Assay for Ly49Q Protein
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The lipid binding ability of Ly49Q was examined using Sphingo Strips (Echelon Biosciences Inc.) according to the manufacturer’s instructions. In brief, Shingo Strip membranes were incubated with GST-tagged Ly49Q proteins or control GST [5 µg/ml in tris-buffered saline (TBS) containing 3% bovine serum albumin (BSA)] at 4°C overnight. After washing with TBS-T to remove unbound GST-tagged proteins, Shingo Strip membranes were further incubated with anti-GST antibody (Santa Cruz Biotechnology, Inc.) and subsequently HRP-conjugated anti-mouse IgG (Jackson Laboratory). HRP was detected with Supersignal West Femto or Supersignal West Pico chemiluminescent substrates with the LAS-3000 detection system (FUJIFILM Co., Tokyo, Japan).
4
Quantitative Analysis of RyR2 and MG23 in Cardiac Tissue
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HSR proteins or H9C2 cell lysates were size-fractionated by SDS-PAGE on a 4–12% Bis-Tris precast gel (Life Technologies). After separation, proteins were transferred to a nitrocellulose membrane, and nonspecific binding sites were blocked for 1 h at room temperature using 5% dried milk and Tris-buffered saline (0.1% Tween 20, pH 7.4). Membranes were probed overnight at 4 °C with primary antibodies specific for RyR2 phosphorylated at site serine 2809 (1:5000, Badrilla, Leeds, UK), anti-MG23 (1:20,000 Sigma), and anti-β-actin (1:10,000, Sigma) in 5% dried milk and Tris-buffered saline. Secondary horseradish peroxidase-linked goat anti-rabbit or anti-mouse IgG (both 1:10,000) Sigma) antibodies were used in combination with an enhanced chemiluminescent detection system (Thermo Scientific, Paisley, UK) and visualized using a Fujifilm LAS-3000 detection system. Densitometry was performed using ImageJ software (National Institutes of Health).
5
Western Blot Analysis of Signaling Proteins
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Cells were washed with cold phosphate-buffered saline (PBS) and harvested with radioimmunoprecipitation assay lysis buffer containing phosphatase inhibitor cocktail set V (#524629; MERCK, Kenilworth, NJ, USA) and protease inhibitor cocktail set III (#535140; MERCK). Whole-cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were blotted onto polyvinylidene fluoride membranes (#10600030; Amersham, Little Chalfont, UK). After blocking with 5% skim milk in Tris-buffered saline buffer (pH 8.0) with 0.1% Tween-20, the membrane was incubated with the primary antibody overnight (Cell Signaling Technology, Danvers, MA, USA; anti-pEGFR: #3777S; anti-EGFR: #4267S; anti-pMET: #3077S; anti-MET: #8198S; anti-pAKT: #9271S; anti-AKT: #9272S; anti-pERK: #4370S;anti-ERK: #4695S; anti-E-CADHERIN: #3195S; anti-VIMENTIN: #5741S). After rinsing with Tris-buffered saline buffer, the membrane was incubated with a secondary antibody for 1 h and washed, followed by visualization using electrochemiluminescence (SuperSignal™ West Femto Maximum Sensitivity Substrate; Thermo Fisher Scientific; #34095) and a LAS-3000 detection system (Fujifilm, Tokyo, Japan).
6
Western Blotting for Protein Analysis
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Western blotting was performed as previously described [20 (link)]. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Elpis-biotech, Daejeon, Republic of Korea), and proteins were detected by enhanced chemiluminescence (Advansta, San Jose, CA, USA) using the LAS 3000 detection system (Fuji Film, Tokyo, Japan). Primary antibodies used were FLG (1:500; LifeSpan BioSciences, Seattle, WA, USA); IVL (1:500; BioLegend, San Diego, CA, USA); pYAP, CERB, p38, EKR, JNK, Cyclin B1, D1, E1, CKD2 (1:1000; Cell Signaling, Beverly, MA, USA); MMP-3, COL1A1, YAP, TYRP-1,2, PKA, Cyclin A, β-actin (1:500; Santa Cruz Biotechnology, Dallas, TX, USA); MC1R (1:500; Bioss Antibodies, Woburn, MA, USA); MITF, TYR, EDN1, WNT, and CDK1 (1:1000; Abcam, Cambridge, UK).
7
Western Blot Analysis of Intestinal Proteins
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Caco-2 cells or colitis mice’s colons were harvested and lysed with RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) and 1 mM PMSF, protease inhibitor cocktail. Protein concentration was measured and equal amounts of proteins were loaded and separated on SDS-PAGE (EBA-1041, Elpis Biotech, Korea), and transferred to a polyvinylidene difluoride membrane (PVDF, IPVH00010, Sigma Aldrich). The membranes were incubated with antibodies for ZO-1 (Abcam, Cambridge, UK), Occludin, β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), E-cadherin, p-P38, p38, p-ERK1/2, and ERK1/2 (Cell Signaling Technology, Danvers, MA, USA). Proteins were detected by enhanced chemiluminescence (K-12045-D-50, Advansta, San Jose, CA, USA) and visualized using the LAS 3000 detection system (Fujifilm Corporation, Tokyo, Japan).
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