In total, 20 µg of rDer f 1/2 fusion protein was loaded in each well. Proteins were separated by 12% SDS-PAGE and then transferred to polyvinylidene difluoride membranes for western blotting. For dot blotting, 2 µl of the allergens (rDer f 1, rDer f 2 and rDer f 1/2) was separately spotted onto nitrocellulose membranes at a concentration of 1 µg/µl. The membranes were blocked with 5% Difco™ Skim Milk (DSM; BD Biosciences, San Jose, CA, USA) diluted in TBS containing 0.05% Tween 20 (TBST) at 4°C overnight. Serum samples from 15 HDM-allergic patients and 15 control subjects were also analyzed and incubated for 2 h at 37°C. Subsequently, mouse anti-human IgE Fc-HRP antibody was applied (1:2,000 in TBST with 1% DSM) for 1 h at 37°C. Antigen-antibody complexes on membranes were visualized using a Pierce™ DAB kit.
Difco skim milk dsm
Manufactured by BD
Sourced in United States
Difco™ skim milk (DSM) is a dehydrated, fat-free milk powder used as a culture medium component in microbiological applications. It provides essential nutrients for the growth and maintenance of microorganisms in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Microplate absorbance reader, by Bio-Rad (1 mentions)
Mouse anti human ige horseradish peroxidase conjugated antibody, by Southern Biotech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Pierce 3 diaminobenzidine substrate kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using difco skim milk dsm
1
Immunoblot Analysis of Der f Allergens
2
Western Blot and Dot Blot Analysis
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Purified proteins were separated by 12% SDS-PAGE and then transferred to polyvinylidene-fluoride membranes (Millipore, USA) for western blotting. For dot blotting, 2-µg aliquots of polypeptides were applied serially onto nitrocellulose membrane (Millipore, USA); BSA was used as a control. The membranes were blocked with 5% Difco™ skim milk (DSM, BD Biosciences, USA) diluted in Tris buffered saline containing 0.05% Tween 20 (TBST) at 4 °C overnight. Serum samples were incubated for 2.5 h at 37 °C, and then incubated with horse radish peroxidase-conjugated mouse anti-human IgE antibody (1:2000 in TBST with 1% DSM) for 1 h at 37 °C. Antigen–antibody complexes on membranes were visualized with a Pierce™ 3′-diaminobenzidine substrate kit.
3
IgE-ELISA Assay for Protein Quantification
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The IgE-ELISA assay developed in this study was performed as previously described.33 (link) Microtiter plates were coated with recombinant polypeptides (200 ng/well) in 0.1 mol/L and pH 9.2 carbonate buffered solution (Leagene, Beijing, China) at 4 °C overnight. The samples were blocked with 300 μl 5% (w/v) Difco™ skim milk (DSM; BD Biosciences) in phosphate-buffered saline containing 0.05% Tween 20 (PBST) at 37 °C for 3 h. Serum samples (1:10 dilution in 1% DSM-PBST) were added to each well (100 μL/well) and incubated for 2.5 h at 37 °C. The plates were incubated with mouse anti-human IgE horseradish peroxidase-conjugated antibody (#9160-05; 1:2000 dilution; Southern Biotech) for 1.5 h at 37 °C. Each incubation step was followed by five washes with PBST. Binding was detected with 100 μl of 1-mM 3,3,5,5′-tetramethylbenzidine substrate (Invitrogen; Thermo Fisher Scientific); the substrate reaction was stopped with 50 μl 2 M H2SO4 per well. The plates were read by an absorbance microplate reader (Bio-Rad, USA) at 450 nm. IgE-ELISA results with a positive/negative result sample optical density ratio value > 2.1 were considered positive. All tests were performed in triplicate.
4
Immunoblotting analysis of rDer f 24 allergen
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rDer f 24 and hybrid proteins were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore) for western blotting. For dot blotting, 2 μl rDer f 24 (concentration 1 μg/μl) and synthetic polypeptides (BSA-1-32 aa, BSA-23-54 aa, BSA-45-76 aa, BSA-67-98 aa and BSA-89-118 aa) were dotted onto PVDF membranes. The membranes were blocked with 5% Difco™ skim milk (DSM; BD Biosciences) diluted in Tris buffered saline containing 0.05% Tween-20 at 4°C overnight. Western blot membranes were incubated with a mixture of allergic sera from different 10 patients with HDM allergy (1:10 dilution) for 2 h at 37°C. For dot blotting, a mix of 10 serum samples from HDM-allergic patients with high IgE-binding to rDer f 24 and a mix of 3 non-allergic sera samples (1:10 dilution) were incubated for 2 h at 37°C. Following washing, the membranes were incubated with horse radish peroxidase-conjugated mouse anti-human IgE antibody (1:2,000 dilution; cat. no. 9160-05; SouthernBiotech) for 1.5 h at 37°C. Antigen-antibody complexes on membranes were visualized using a Pierce™ 3'-diaminobenzidine substrate kit (Thermo Fisher Scientific, Inc.).
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