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Antibodies and Antisera
Published in Lars-Inge Larsson, 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.
Relation of Antigliadin Antibodies to Gluten-Sensitive Enteropathy
Published in Tadeusz P. Chorzelski, Ernst H. Beutner, Vijay Kumar, Tadeusz K. Zalewski, Serologic Diagnosis of Celiac Disease, 2020
Wim Th. J. M. Hekkens, Marja van Twist - de Graaf
The method is useful for the blotting of small proteins, but blotting of larger proteins is time-consuming, resulting in broadening of the protein bands. Apart from this, the procedure is not quantitative in contrast to the electroblotting. The principle of this technique is that proteins are blotted by placing the gel/membrane assembly in an electric field perpendicular to the plane of the gel.96 The proteins are thus driven out of the gel onto the membrane. The equipment consists either of a tank with platinum electrodes in which one blotting buffer is used or of a semidry blotting apparatus with carbon electrodes; filter paper soaked in electrode buffers is used as a buffer compartment in this case. In this manner different electrode buffers may be used to increase the efficiency of the blotting.
Chemical Cleavage of Peptide Bonds
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
Xu and Shively17 have described improvements on the electroblotting of proteins. These investigators reported higher degrees of success with polyvinyldifluoride (PVDF) membranes. Transfer yields were markedly improved upon pretreatment of the membranes with Polybrene.
Hypoxia and acidity regulate immune checkpoint molecule and IFN-β expression in non-small cell lung cancer cell lines
Published in Journal of Receptors and Signal Transduction, 2023
Achilleas G. Mitrakas, Alexandra Giatromanolaki, Michael I. Koukourakis
Whole extracts from cell lines were isolated in a sucrose lysis buffer (0.25 M sucrose and 25 mM Tris-HCl) after different incubations and then homogenized using a plastic pestle. The protein concentration of each sample was calculated using the BCA Protein Assay Kit (Thermo Fisher Scientific), and 30 μg of protein was loaded for analysis. Protein samples were separated on discontinuous SDS gels using a 10% separating gel and 5% stacking gels. Electroblotting was performed with Porablot PVDF membranes (Macherey Nagel, Germany). Then membranes were blocked with 5% BSA in 150 mM NaCl, pH 7.5 10 mM Tris (TBS), and 0.1% (v/v) Tween 20 at 20–25 °C for 2h followed by overnight hybridization with different antibodies at 4 °C. Analytically, recombinant anti-CD80 antibody [EPR1157(2)] (1:500, ab134120, abcam, UK), anti-CD47 antibody [CD47/2937] (1:1.000, ab260418), anti-Interferon beta antibody (1:1.000, ab85803, abcam, UK), anti-LDHV, (1:1000, ab9002, abcam, UK), and anti-PD-L1 [CAL10] (1:500, Biocare, CA, USA).
Selective recognition of human small transthyretin aggregates by a novel monoclonal antibody
Published in Amyloid, 2023
A. C. Teixeira, Maria J. Saraiva
Ten ul of fractionated TTR obtained by SEC were applied in a Native Mini-Protean TGX 4-15% gradient gel (BIO-RAD), under non-denaturing and non-reducing conditions, and electroblotted onto PVDF membrane in a semi-dry iBlot system (Invitrogen). After blocking, immunodetection was performed by using CE11 mAb hybridoma supernatant, diluted 1/20 with blocking buffer, for one hour RT. After incubation with conjugated goat anti-mouse IgG (H + L) HRP (Invitrogen) 1:5000 dilution, TTR was visualised using the enhanced chemiluminescence reagent (ECL). Blots were, subsequently incubated with the polyclonal anti-TTR DAKO without stripping. The molecular weight of the bands was calculated through comparison with a silver-stained Native Mark Unstained Protein Standard (Invitrogen) run in the same gel that was used for electroblotting. After electrophoresis the gel containing marker was cut and stained with silver. The gel and membrane were put side by side and matched to adjust the size of the gel.
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.