7. The antibodies sure seem to be taking a long time. You are dying to know where in the cell YFP is localized (in the cytoplasm? ER? Lysosome? a) Briefly, how could you attempt to determine this before the antibodies are ready? b) Briefly, how could you determine the localization after you get the antibodies?

7. The antibodies sure seem to be taking a long time. You are dying to know where in the cell YFP is localized (in the cytoplasm? ER? Lysosome? a) Briefly, how could you attempt to determine this before the antibodies are ready? b) Briefly, how could you determine the localization after you get the antibodies?

Basic Clinical Laboratory Techniques 6E

6th Edition

ISBN:9781133893943

Author:ESTRIDGE

Publisher:ESTRIDGE

Chapter4: Basic Immunology And Immunohematology

Section4.1: Introduction To Immunology

Problem 10RQ

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Labs that do a lot of Western blots often have hundreds of primary bodies but only two or three secondary antibodies. Why? I understand that secondary antibody aids in the detection, sorting or purification of target antigens by binding to the primary antibody which directly binds to the target antigen. However, I don't undrstand why there are only two or three secondary antibodies available in the laboratory. Glad if the expert would advise.
a) What is “washing” in immunology? What types of interactions doeswashing interfere with? What molecules are used for effective“washing” in the immunology lab?b) Blocking is also a common feature of immunological assays. Why isblocking required?i. A student forgot to block their Western blot membrane beforeadding the secondary antibody. They continue and block themembrane after the secondary antibody step, assuming itdoesn’t matter which order the steps are completed in. Howwould this affect the results seen?ii. A student blocked their ELISA plate with 50 microlitres ofblocking buffer, although their coating, binding and detectionsteps were at 100 microlitres. How would this affect the resultsseen?
5) The immunoblot shows probing of samples taken from a time course after treatment with the proteasome inhibitor MG132. Recall that the proteasome is responsible for selectively degrading only those proteins that have been tagged with ubiquitin. The left indicates the antibody that is used to detect protein for each panel. P53 P27 AGR2 GAPDH MG132 (h) 0 0.5 1 2 4 8 12 24 Image from Wang et al., (2019) Oncogene 38:3458-3474. a) What effect does MG132 have on each protein? What does this tell you about each of these proteins? ● P53 P27 AGR2 GAPDH b) How do we know that the differences we observe in the proteins is not due to un- equal loading of samples into each lane of the gel?
Observe the structure of antibodies below that has four protein subunits. Explain why we only see two (not four) bands on the SDS PAGE gel.
The loopy polypeptide segments at the very top of the structure shown are the segments that actually contact the antigen. Would you expect these binding segments to be rigid or flexible?
Some pathogenic microorganisms encode proteins, such as the Staphylococcus Protein A, that bind to immunoglobulin constant region domains with high affinity. These microbial proteins provide a benefit to the microorganism by: Preventing antibodies bound to the microbe from binding to Fc receptors on phagocytes Blocking the binding of anti-microbial antibodies to the pathogen surface Cleaving the antibody into fragments that separate the antigen-binding region from the effector function Inducing aggregation of the anti-microbial antibodies by multivalent binding to the pathogen-derived protein Preventing the antibody from neutralizing the pathogen
refer to the picture explain in detail the type of centrifugation which you will use to seperate imunoglobulin M and immunoglobulin G
Several vaccines against viral infections are made by isolating purified surface proteins of the viral particle, mixing them with an adjuvant to stimulate an innate immune response, and injecting the mixture into people. Two examples of this are the vaccine against Hepatitis B virus, and the vaccine against Human Papilloma Virus (the ‘cervical cancer’ vaccine). One interesting property of vaccines of this type (known as ‘subunit vaccines’) is that there is a requirement for a CD4 T cell response to the vaccine antigen in order to generate antibodies to the innocuous protein in the vaccine. In the case of the Hepatitis B vaccine, the viral protein included in the vaccine is the Hepatitis B surface antigen (HepB-SAg), a protein that is approximately 200 amino acids in length. The graph in Figure Q4.27 shows the data from immunizing individuals with this vaccine, and monitoring their production of protective antibody responses to the viral protein. a) What results would be predicted if…
Antibody binding to a pathogen surface is greatly enhanced when both antigen-binding sites of the antibody are engaged at once, a feature known as bivalent binding. It is possible for antibodies to bind bivalently to a wide variety of components on many different pathogen surfaces due to the flexibility in the protein at the hinge region and at the V–C junction.
a) How virus vaccine, under immune system processes (via dendritic cells, T-Cells and B-Cells), can help people against real virus infection. b) Structure of SARS-CoV-2 and their entry process on host cells. Then, if you choose onetype of virus protein (spikes, nucleocapsid, membrane or envelope) should be used for vaccine,which is the best candidate? Why?
You are interested in performing indirect immunofluorescence light microscopy to observe the localization of the catalase enzyme in the cultured HeLa cells, obtained historically from the cervical tumor of Henrietta Lacks. You were going through the lab stock and found a few primary and secondary antibodies. Which of the following secondary antibody can you use in your experiments? O All of the mentioned antibodies can be used in the experiment Goat anti-human antibody conjugated to 10 nm gold Goat-anti-human catalase conjugated to 10 nm gold O Human anti-catalase antibody conjugated to fluorescent rhodamine Goat anti-human antibody conjugated to fluorescent rhodamine
2. https://doi.org/10.1186/s12868-022-00692-1 (link to research) a) In the immunohistochemistry section of the materials and methods section the authors wrote “The number of positive cells in hotspot areas in ten high power fields (HPFs) in areas of demyelination and plaques in the brain stem were counted using the image analysis software (Lecia Application Suite Version 4.12.0, Welzlar, Germany).” Why were they looking at demyelination areas for this study? b) In the effect of mitoxantrone on histopathological changes in the brain section of the results section the authors wrote “Active plaques revealed inflammatory cellular infiltrates with abundant macrophages stuffed with myelin debris, an evidence of ongoing myelin breakdown.” What does macrophages stuffed with myelin debris have to do with the study?