Strain UMI-01 was cultured in the minimum salt medium containing 2% (w/v) glucose or 1% (w/v) alginate as the sole carbon source at 25 °C. When the OD600 of the culture medium was between 0.6 and 0.8, the cells were harvested by centrifugation at 12,000g for 15 min at 4 °C, and soluble proteins were obtained using BugBuster Master Mix (Merck Millipore, Burlington, MA, USA) according to the manufacturer’s protocol. Each sample (5 µg) was subjected to SDS-PAGE and electroblotted onto a ClearTrans nitrocellulose membrane (FUJIFILM Wako Pure Chemical Corporation). The anti-FlRed, -FlKin, -FlAld, -FlDet, and -FlDeg antibodies were raised in rabbits against the synthetic peptides CKGAERVAELAAEGI, CYGLNEEPLDNQRAL, CSKLIKPESDGNFDL, CLVMDSRKDPRAASA, and CDTAQPNRTPLPKSD, respectively, using keyhole limpet hemocyanin as a carrier protein (Eurofins Genomics, Tokyo, Japan). Each antibody was purified by affinity chromatography using an immobilized antigen column (Cellufine Formyl, JNC Corporation, Tokyo, Japan) and used as the primary antibody. A horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich) was used as the secondary antibody, and signals were detected using Western BLoT Quant HRP Substrate (TaKaRa).
Cleartrans nitrocellulose membrane
Manufactured by Fujifilm
Sourced in Japan
The ClearTrans Nitrocellulose Membrane is a laboratory product manufactured by Fujifilm. It is a thin, porous membrane made of nitrocellulose material. The primary function of this membrane is to facilitate the transfer and immobilization of biomolecules, such as proteins or nucleic acids, during analytical procedures.
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Lab products found in correlation
Mouse anti glyceraldehyde 3 phosphate dehydrogenase monoclonal antibody, by Fujifilm (2 mentions)
Hrp conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (2 mentions)
Western blot quant hrp substrate, by Takara Bio (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg antibody, by Merck Group (1 mentions)
Bugbuster master mix, by Merck Group (1 mentions)
Imagequant 800, by GE Healthcare (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Las 3000 luminescent image analyzer, by Fujifilm (1 mentions)
Can get signal immunoreaction enhancer solution, by Toyobo (1 mentions)
Anti cd81 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti β actin mouse monoclonal antibody, by Fujifilm (1 mentions)
Bz x700, by Keyence (1 mentions)
5 protocols using cleartrans nitrocellulose membrane
1
Protein Expression Analysis of Strain UMI-01
2
Immunoblotting: A Comprehensive Protein Analysis
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Immunoblotting was performed as previously described (13 (link)). Briefly, cell lysis buffer containing 50 mM Tris-HCl (pH 8.0), 5 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0), 5 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton X-100, 1 mM Na3VO4, 20 mM sodium pyrophosphate and Roche's complete protease inhibitor cocktail. The protein concentrations were measured by DC protein assay kit (Bio-Rad Laboratories, Inc.), and were separated by SDS-PAGE, then transferred onto ClearTrans Nitrocellulose Membrane (Wako). Membranes were blocked with 0.5 or 3% skim milk, and treated with primary antibodies in Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBS-T). After treating with secondary antibodies in TBS-T, immunoreactive bands were detected using ECL Pro Western Blotting Detection Reagent (PerkinElmer.) and visualized using a LAS-3000 luminescent image analyzer (Fujifilm) or ImageQuant 800 (GE Healthcare). Valosin-containing protein (VCP) was used as a loading control.
3
Western Blot Analysis of Protein Lysates
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Cells (4 × 106) were lysed by 200 μl of 4xSDS-PAGE sample buffer (containing 0.5 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, and 5 μg/ml aprotinin) with/or without 1% 2-mercaptoethanol. When CD81 was detected by Western blotting with anti-CD81 antibody (Santa Cruz Biotechnology, TX, USA), the sample buffer without 2-mercaptoethanol was used. The sample was boiled for 5 min and sonicated for 30 s with a probe type sonicator in order to shear the chromosomal DNA. The resulting lysate was subjected to SDS-PAGE on 8 or 12% polyacrylamide gel followed by Western blotting38 (link),39 (link). The proteins were transferred onto a ClearTrans® nitrocellulose membrane (Wako, Osaka, Japan), and the membrane was incubated with 3% non-fat dry milk in PBS containing 0.1% Tween-20 (PBS-T) for 1 h at room temperature. Then, the membrane was incubated with a primary antibody and subsequently with a secondary antibody (horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibody) in Can Get Signal Immunoreaction Enhancer Solution (Toyobo, Tokyo, Japan). Primary antibodies used in these experiments included those against FLAG(DDDDK)-tag (MBL, Nagoya, Japan) and CD81, Pax5, Pax2, Pax8 and β-actin (Santa Cruz Biotechnology, TX, USA).
4
Western Blot Analysis of Protein Samples
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We reduced the cell extracts and liver homogenate samples with mercaptoethanol at 95 °C for 10 min. We loaded the samples at 10 μg of protein per lane and then separated them on a 12% polyacrylamide gel. Following the electrophoresis, we blotted the proteins on a nitrocellulose membrane (ClearTrans Nitrocellulose Membrane; Fujifilm Wako Pure Chemical) in a semidry blotting system (NA‐1512S; Nihon Eido, Tokyo, Japan) [20 (link)]. The nitrocellulose membranes were blocked with 2% skim milk. We then incubated the membranes with rabbit anti‐CA3 antibody (1 : 8000; Proteintech, Rosemont, IL, USA), rabbit anti‐catalase antibody (1 : 10,000; produced by our laboratory), mouse anti‐β actin monoclonal antibody (1 : 10,000; Fujifilm Wako Pure Chemical), or mouse anti‐glyceraldehyde‐3‐phosphate dehydrogenase monoclonal antibody (1 : 5000; Fujifilm Wako Pure Chemical). This was followed by incubation with horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit immunoglobulin G (IgG) antibody (1 : 10,000; SeraCare, Milford, MA, USA) or HRP‐conjugated goat anti‐mouse IgG antibody (1 : 2500; Biosource, Camarillo, CA, USA). Finally, we visualized the protein bands using ImmunoStar LD or ImmunoStar Zeta (both Fujifilm Wako Pure Chemical) with a LuminoGraph (Atto, Tokyo, Japan). We subjected the bands to densitometric analysis using imagej (National Institutes of Health, Bethesda, MD, USA).
5
Western Blot and Melanosome Localization in B16 Melanoma Cells
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Cell extracts (10 μg) were reduced with mercaptoethanol at 95°C for 10 min and then separated on a 12% polyacrylamide gel. Proteins were blotted on a nitrocellulose membrane (ClearTrans Nitrocellulose Membrane; Fujifilm Wako Pure Chemical, Osaka, Japan) by using a semi-dry blot system (NA-1512S, Nihon eido, Tokyo, Japan). The membranes were blocked with 2% skimmed milk. The blocked membranes were incubated with rabbit anti-vinculin antibody (1:1,000) or mouse antiglyceraldehyde-3-phosphate dehydrogenase monoclonal antibody (1:5000; Fujifilm Wako Pure Chemical) and then with horseradish peroxidase-conjugated anti-rabbit IgG goat antibody (1:10,000; BioSource International) or HRP-conjugated goat anti-mouse IgG antibody (1:2500; BioSource International). The protein bands were subsequently visualized with a ImmunoStar Zeta by using a luminograph apparatus (Atto, Tokyo, Japan). 2.6. Microscopic observation of melanosome localization in B16 melanoma cells B16 cells were seeded onto a 6-well plate (TrueLine, San Jose, CA). After incubation for 24 h, the cells were exposed to UVA at 20 W m -2 by using the 365-nm LED light source for 15 min. At 24 h after UVA irradiation, the distribution of melanosomes in B16 cells were observed using a microscope (BZ-X700, Keyence).
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