Manufacturing and Standardizing Allergen Extracts in Europe
Richard F. Lockey, Dennis K. Ledford in Allergens and Allergen Immunotherapy, 2014
The complexity of the composition of allergen extracts can be assessed by several techniques. These techniques are standard biochemical and immunochemical separation techniques. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) [44] is a widely used high-resolution technique available in rapid and partly automated systems. The proteins are separated, but only after denaturation, according to size. Densitometric scanning has been reported, but this technique is not quantitative due to differences in staining intensities. It should be used only for a qualitative assessment of the allergen extract. In combination with electroblotting [45], the proteins can be immobilized on protein-binding membranes, such as nitrocellulose, and stained using a variety of dyes or labeled antibodies (immunoblotting), thereby considerably increasing the sensitivity. Some allergens, however, are irreversibly denatured by SDS treatment and may escape detection by IgE immunoblotting [46].
Manufacturing and standardizing allergen extracts in Europe
Richard F. Lockey, Dennis K. Ledford in Allergens and Allergen Immunotherapy, 2020
The complexity of the composition of allergen extracts can be assessed by several techniques. These techniques are standard biochemical and immunochemical separation techniques. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) [32] is a widely used high-resolution technique available in rapid and partly automated systems. The proteins are separated, but only after denaturation, according to size. Densitometric scanning has been reported, but this technique is not quantitative due to differences in staining intensities. It should only be used for a qualitative assessment of the allergen extract. In combination with electroblotting [57], the proteins can be immobilized on protein-binding membranes, such as nitrocellulose, and stained using a variety of dyes or labeled antibodies (immunoblotting), thereby considerably increasing the sensitivity. Some allergens, however, are irreversibly denatured by SDS treatment and may escape detection by IgE immunoblotting [34].
Antibodies and Antisera
Lars-Inge Larsson in Immunocytochemistry: Theory and Practice, 2020
From these considerations, it is evident that the interpretative problems with this technique are great when it is being used as a support for immunocytochemical results. Hence, this technique can be used to verify the presence of an antigen of the same apparent molecular size and isoelectric point as that sought in a tissue. However, it cannot be used to exclude other cross-reactive antigens (e.g., those of much lower molecular weight) from being present. Again, the issue is bedeviled by the fact that neither immunocytochemical nor electroblotting methods are looking at native antigens. In the tissue-staining studies, fixation may destroy the reactivity of the antigen sought, but preserve reactivity of a cross-reactive antigen. The antigen sought may turn up on the immune-blotting replica, but the cross-reactive antigen may escape detection (Figure 6). Accordingly, a totally erroneous interpretation of the results may ensue. The situation is improved (but not completely rectified) by model studies, showing that the tissue fixation and other maneuvers introduced immunocytochemically do not affect the antigenicity of the molecule sought. If necessary, such studies can be conducted directly on the electroblotting replica.
Antagonist of cIAP1/2 and XIAP enhances anti-tumor immunity when combined with radiation and PD-1 blockade in a syngeneic model of head and neck cancer
Published in OncoImmunology, 2018
Roy Xiao, Clint T. Allen, Linda Tran, Priya Patel, So-Jin Park, Zhong Chen, Carter Van Waes, Nicole C. Schmitt
For assessment of IAP levels, MOC1 cells were cultured in 6-well plates in complete media with or without ASTX660 for 24 hours. For flow cytometry, cells were then harvested, fixed in 2% PFA, and permeabilized overnight with methanol at −20°C. Cells were then rinsed and stained with an Alexa-647 conjugated antibody for XIAP or a primary antibody for cIAP2 followed by Alexa-488-conjugated secondary antibody, then analyzed by flow cytometry. For western blot, whole-cell lysates were obtained with NP40 lysis buffer. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific). Samples were then mixed with NuPAGE LDS sample buffer and NuPAGE sample reducing agent (Life Technologies), heated at 95°C for 5 minutes and subjected to electrophoresis using 4%–12% Bis-Tris precast gels (Life Technologies) at 150 V for 75 minutes. Proteins were then transferred by wet electroblotting onto a nitrocellulose membrane. Membranes were blocked for one hour in Odyssey blocking buffer (LI-COR Biosciences) and incubated with the primary antibody overnight at 4°C, then rinsed and incubated with species-appropriate horseradish peroxidase (HRP)-conjugated antibody for one hour. Blots were incubated with Chemiluminescent HRP Antibody Detection Reagent (Denville Scientific Inc.) and imaged using Image Studio software (LI-COR Biosciences).
Bacopa monniera extract mitigates isoproterenol-induced cardiac stress via Nrf2/Keap1/NQO1 mediated pathway
Published in Archives of Physiology and Biochemistry, 2022
T. Mohan Manu, T. Anand, G. R. Sharath Babu, Mahantesh M. Patil, Farhath Khanum
Expression levels of Bcl2, Bax, Nrf2, Keap1, NQO1,HO-1 and NOS2 were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Ten percent heart homogenate was prepared in lysis buffer, pH 7.4 and total protein levels were measured by the method of Lowry et al. (1951) using BSA as standard. Tissue homogenates containing 150 µg of proteins were separated on SDS-PAGE and transferred onto a nitrocellulose membrane using an electroblotting apparatus (Cleaver Scientific Ltd, UK). Proteins transferred onto membranes were blocked with 5% milk in TBST for 3 h at 4 °C. Membranes were probed with primary antibodies of GAPDH (sc-5286), Bcl2 (sc-8637), Bax (sc-34280), Nrf2 (sc-722), NQO1 (sc-16464), Keap1 (sc-15246), hemeoxygenase-1(sc-10789) and NOS2 (sc-651) (Santa Cruz Biotechnology, CA, USA) at 1: 1,000 dilutions in TBST with 5% milk and incubated overnight at 4 °C. The membranes were washed with TBST for three times at 5 min interval, followed by incubation at room temperature for 2 h in horseradish peroxidase-conjugated goat anti-mouse or rabbit anti-goat or mouse anti-rabbit secondary antibodies (DAKO, Denmark) at 1: 10,000 dilutions. The membranes were washed and developed using a chemiluminescence detection system (ProteoQwest®, Sigma). Developed bands on membranes were captured by exposure to X-ray film. The intensity of the bands on X-ray film was measured using NIH ImageJ software.
Trefoil factor 3 in perinatal pancreas is increased by gestational low protein diet and associated with accelerated β-cell maturation
Published in Islets, 2018
Louise Winkel, Annika Bagge, Louise Larsen, Tobias N. Haase, Morten Rasmussen, Jeanette Lykke, Dennis B. Holmgaard, Lars Thim, Jens H. Nielsen, Louise T. Dalgaard
INS-1E cells were lysed in RIPA buffer as described previously.36 One milligram of protein extracts were incubated with antibody as indicated overnight at 4°C. Complexes were precipitated using Dynabeads M-280 sheep anti-rabbit or anti-mouse IgG according to instructions from the manufacturer. The antigens were eluted by incubation for 5 min at 95°C in RIPA buffer containing 0.11 μM β-mercaptoethanol and 1x NuPage LDS sample buffer (Invitrogen). Proteins were separated by electrophoresis and transferred to nitrocellulose membranes by electroblotting. After blocking the membrane was exposed to primary antibody overnight at 4°C (anti-EGFR, anti-β-catenin, anti-phosphotyrosine and subsequently washed in Tris-buffered saline containing 0.1% Tween20, incubated with peroxidase-conjugated secondary antibody and proteins detected by chemiluminescence using ECL plus Western blotting detection reagent (GE Healthcare). Following phosphotyrosine blotting, blots were stripped (Pierce stripping buffer) and exposed to EGFR or β-catenin antibody to ascertain equal loading. Blots were quantified using Fiji (https://fiji.sc/),37 controlled for loading differences and normalized to the control condition.