S. mutans cells and cell wall preparations were each resuspended in PBS to reflect original cell culture volumes. For each sample, 200 μL of serial two-fold dilutions in PBS, beginning at 1:8, were applied to a PBS-soaked nitrocellulose membrane under vacuum using a 96-well manifold dot blot apparatus (Minifold I; Whatman). Membranes were blocked for 1 h at room temperature with PBS containing 0.2% Tween-20 (PBS-Tw), then reacted with P1 specific monoclonal antibodies (MAbs) obtained from previously established hybridomas (Ayakawa et al. 1987 (link)). MAbs included 6–8C, which reacts with P1’s C2 and C3 domains; 4–10A, which reacts with the hybrid helical stalk; and 1–6F, which reacts with the globular head domain (Heim et al. 2015 (link)). Anti-P1 MAbs were used as ascites fluids diluted 1:1000 in PBS-Tw. Following overnight incubation at room temperature, the membranes were washed four times for 15 min with PBS-Tw, reacted for 2 h with horseradish peroxidase-labeled goat-anti-mouse secondary antibody (ICN/Cappell Biomedicals) diluted 1:1000 in PBS-Tw, washed twice with PBS-Tw and twice with PBS, then developed with 4-chloro-1-naphthol solution (7 mL PBS, 1 mL 4-chloro-1-naphthol [Sigma, 3 mg/ml in ice cold methanol], and 8 μL of 30% hydrogen peroxide).
4 chloro 1 naphthol
Manufactured by Merck Group
Sourced in United States, Germany
4-chloro-1-naphthol is a chemical compound used in various laboratory applications. It is a white to light yellow crystalline solid. 4-chloro-1-naphthol is commonly used as a detection reagent in biochemical and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
0.45 m nitrocellulose membrane, by Bio-Rad (2 mentions)
Bio1d software, by Vilber (2 mentions)
Hrp conjugated goat anti rabbit igg antibody, by Merck Group (2 mentions)
Hrp conjugated anti mouse igg, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Horseradish peroxidase conjugated anti human igg, by Merck Group (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Mini protean, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Hybond c extra, by Cytiva (1 mentions)
Isopentane, by Merck Group (1 mentions)
Biotinylated goat anti mouse igg, by Vector Laboratories (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Bio dot microfiltration apparatus, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated streptavidin, by Merck Group (1 mentions)
Ez link sulfo nhs lc biotinylation kit, by Thermo Fisher Scientific (1 mentions)
31 protocols using 4 chloro 1 naphthol
1
Dot blot immunoassay for S. mutans P1 protein
2
Western Blot Analysis of Immunized Sera
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SDS-PAGE was performed as described above. Following the separation, proteins were transferred to a 0.45 µm nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA) in a transfer buffer (25 mM Tris, 192 mM Glycine, Methanol 20%, pH 8.3) over a period of approximately 24 h at 50 V. Strips containing approximately 10 µg of protein were blocked with nonfat milk 5% (La Sereníssima), overnight, at 4 °C. Afterwards, membranes were incubated for 2 h with pooled pre-immune or immune sera of mice collected after one (OMV, OMV+AH and OMV+DDA) or two (OMV+Sap) SC boosters, at a dilution of 1:50, at RT. Then, HRP-conjugated anti-mouse IgG (γ chain) (1:10,000) (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) was incubated for 2 h, at RT. The enzymatic reaction was developed with 4-chloro-1-naphthol (Sigma-Aldrich, San Louis, MO, USA) and was stopped with distilled water after 20 min.
3
Dot Blot Assay for LPS Antibody Detection
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The dot blot assay was carried out with a panel of P. mirabilis LPS and the synthetic antigen, Lys-GalA. LPSs and Lys-GalA (10 µg/mL) were dotted on a polyvinylidene difluoride (PVDF) (Merck, Darmstadt, Germany) membrane and air dried. The membranes were washed three times Tris-buffered saline (TBS), blocked with 2% bovine serum albumin, BSA (Sigma-Aldrich) in TBS at 37 °C for 1 h, and then washed again three times with TBS. The membrane was incubated with patient sera diluted 1:1000 (v/v) in 1% BSA TBS for 1 h at 37 °C. The membranes were washed three times with TBS. Next, the membrane was incubated with horseradish-peroxidase-conjugated anti-human IgG (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:1000 (v/v) at 37 °C for 1 h and washed with TBS. The membrane was further developed with the chromogenic substrate, 4-chloro-1-naphthol (Sigma-Aldrich, USA) and H2O2 (Sigma-Aldrich). Reaction with rabbit sera anti-LPS O3 was used as positive control. Drops of solvent without antigen were used as negative controls. The PVDF membrane was air dried and a grey-scale digital image was made with a transilluminator and saved in TIFF format. The image was then imported to ImageJ (NIMH) software. The image, with 256 grey levels, was enlarged to 135% to facilitate measurement [11 (link)].
4
Immunoblotting Analysis of Truncated Glycoprotein E
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Centrifugation of E. coli cells that produce truncated analogous of glycoprotein E was performed after 4 h of cells growth with IPTG. Lysis buffer (50 mM Tris-HCl, pH 6.8, 4% sodium dodecyl sulfate, 200 mM dithiothreitol) was added to cell precipitates, then precipitates were fractionated by 12.5% SDS-PAGE and transferred to nitrocellulose membrane. Skim milk diluted in PBST was used as a blocking agent. Then, membranes were incubated at 37 °C for one hour with mAb FVN-32 (5 μg/mL); incubation with anti-TBE mice sera was used as a positive control. Goat anti-mice IgG conjugated with horse-radish peroxidase (Sigma Aldrich, St. Louis, MO, USA) was used to reveal antigen–antibody complexes formed. 4-Chloro-1-naphthol (Sigma-Aldrich, St. Louis, MO, USA) was applied to visualize the complexes.
5
SDS-PAGE and Western Blot Analysis of Anisakis Antigens
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SDS-PAGE analysis of A. simplex antigens and WB reactivity of antigens with rabbit anti-A. simplex serum were performed as described previously [40 (link),72 (link)] using 4-chloro-1-naphthol (Sigma, St. Louis, MO, USA) as a substrate for horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibodies (Sigma, St. Louis, MO, USA).
The molecular weight of the SDS-PAGE and WB bands and densitometric plots of their profiles were estimated using the Bio-1D software (Vilber Lourmat, ver. 15.07, Marne-la-Vallée, France). The ImageJ software (National Institutes of Healths, ver. 1.53a, Bethesda, MD, USA) was used for densitometric calculations of the SDS-PAGE and WB profiles.
The molecular weight of the SDS-PAGE and WB bands and densitometric plots of their profiles were estimated using the Bio-1D software (Vilber Lourmat, ver. 15.07, Marne-la-Vallée, France). The ImageJ software (National Institutes of Healths, ver. 1.53a, Bethesda, MD, USA) was used for densitometric calculations of the SDS-PAGE and WB profiles.
6
Western Blot Analysis of Camel Sera
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After another electrophoresis, under reducing condition in 10% SDS-PAGE, eluted fractions, crude HCF antigens, and Prestained Protein Ladder (Vivantis Technologies) were blotted onto nitrocellulose membrane as described by Towbin et al. [28 (link)] in a blotting system. After washing and blocking, the membrane was incubated with diluted naturally infected camel sera (1:200). Protein A horseradish peroxidase conjugate was used at a dilution of 1:2000. In addition, bands were observed by adding 4-chloro-1-naphthol (Sigma), and the membrane was photographed by Molecular Imager Gel Doc™ XR+ with Image Lab Software.
7
Immunodetection of P67 Protein
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Western blot was performed by separating the purified recombinant protein on 12% SDS-PAGE and transferring it onto nitrocellulose membrane (NCM) by applying current at 0.8 mA/cm2. After overnight blocking with 3% skimmed milk, P67 protein present on the membrane were then immune stained by first exposing the membrane to hyper immune sera raised against M. leachii in calves (positive control) and known negative bovine sera confirmed by both DID and IHA) at a dilution of 1:100 followed by species specific secondary antibodies conjugated to horseradish peroxidase at a dilution of 1:5000 and finally the reaction was developed with 4-chloro-1-naphthol (Sigma) and H2O2. Similarly, dot blot analysis was performed using hyper immune sera as a positive control and known negative sera as a negative control by following the protocol used in western blot after charging the NCM with purified P67 protein.
8
SDS-PAGE and Immunoblotting of Protein Samples
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The protein samples were heated at 95°C for 10 minutes with an equal volume of 2× sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 10% 2-mercaptoethanol, 20% glycerol) and separated by SDS-PAGE in 12% gel [50] (link). For immunoblotting, the separated proteins were transferred to nitrocellulose membranes according to the instructions for the electroblotting apparatus (Mini-PROTEAN, Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked for 1 hour in TTBS (TBS with 0.05% Tween-20) with 1% BSA, washed with TTBS, and incubated overnight at 4°C with purified Abs diluted 1∶1000. After washing with TTBS, the membranes were incubated for 1 hour with goat anti-rabbit Abs conjugated with peroxidase (Bio-Rad Laboratories) diluted 1∶4000 in TTBS, consequently washed with TTBS and TBS, and the peroxidase reaction was performed with 4-chloro-1-naphthol (Sigma-Aldrich, St Louis, MO, USA) as a substrate.
9
Immunoblotting for Dog Sera Analysis
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Immunoblotting was carried out as previously described and adapted to test dog sera (Caballero-Ortega et al., 2008 (link)). In brief, T. gondii proteins from 20 million tachyzoites of the RH strain (type I) were separated by electrophoresis. The proteins were electrotransferred to a nitrocellulose membrane, strips of 3 mm width were cut, and serum samples diluted 1:200 in PBS-T20 were added. Then, goat anti-Dog IgG H&L (HRP) diluted 1:2000 in PBS-T20 was added. Immune complexes were detected using a substrate/chromogen solution with 4-chloro-1-naphthol (Sigma-Aldrich, MA, USA). Sera were considered positive when at least 3 diagnostic bands were detected on the nitrocellulose membrane.
10
Periplasmic Expression of Influenza H5 HA1
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Periplasmic expression of the H5 HA1 gene in JOL1814 was confirmed by Western blot assay. To express H5 HA1 protein, JOL1814 was cultured in Luria–Bertani (LB) broth (Becton, Dickinson and Company, USA) until the OD600 reached 0.8, after which the pellet was harvested by centrifugation at 3400g for 20 min. The periplasmic protein fraction was prepared from harvested cell pellets by the lysozyme-osmotic shock method, as described elsewhere [17 (link), 18 (link)]. Boiled protein samples from the pellet and periplasmic fraction were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, USA). The membranes were blocked with 3% bovine serum albumin. Mouse influenza A avian H5N1 hemagglutinin (HA) (bird flu) polyclonal antibody (Cat. No. MBS396043; MyBioSource, USA) and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgY antibodies (Cat. No. NB730-H; Novus Biologicals, USA) were used as primary and secondary antibodies, respectively. The Western blots were developed by adding 3,3′-diaminobenzidine and 4-chloro-1-naphthol (Sigma) in the presence of H2O2.
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