Antibodies & Cell Signaling: Investigating Protein Phosphorylation

This report explores the application of antibodies in studying cell signaling pathways, focusing on changes in protein phosphorylation and sub-cellular localization. It details how antibodies, specifically immunoglobulins, are crucial for identifying and neutralizing foreign objects and how they play a significant role in understanding cell signaling, a process vital for cellular response to environmental changes. The report discusses various cell signaling pathways, such as HER2 and STAT signaling, and how Rockland antibodies aid in researching these pathways. It also covers the use of phosphorylation-specific antibodies in techniques like multiplexed bead arrays and high-throughput arrays, highlighting their advantages and limitations. Furthermore, the report investigates antibody-based assays, including ELISA and Luminex technology, for protein localization, particularly in response to radiation exposure, emphasizing the importance of sample preparation and antibody quality. The study concludes by underlining the significance of protein transportation and localization in cellular function and discusses methods for tracking extracellular protein levels using finger-prick blood samples or mouth swabs. Desklib offers more resources like this to aid students in their studies.

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TABLE OF CONTENTS
1. Use of antibodies in the study of cell signalling pathways that involve changes in protein
phosphorylation...........................................................................................................................3
2. Using the antibody-based assays to investigate the sub-cellular localisation of a protein......4
REFERENCES................................................................................................................................7

1. Use of antibodies in the study of cell signalling pathways that involve changes in protein
phosphorylation.
Antibodies are also referred as immunoglobulin is basically a protein having Y-shape and
is used by the immune system of the body of humans in identifying and neutralizing the effect of
the foreign objects such as the pathogenic viruses and fungi. It has the capability to recognise the
pathogen’s unique molecule known as antigen.
It also plays a major role in the study of the pathways of cell signalling. It is a biochemical
process through which cells are aligned for responding to the anxiety and the disturbance in the
environment. Signalling helps in regulating the most of the processes of the normal cells but if
the signalling is improper then it can lead to various other diseases. In the process of signalling,
there is a participation of various biomolecules such as amino acids, proteins, second messengers
(For example, inositol triphosphate) and lipids. The main aim here is signalling regulation
through the method of protein phosphorylation which contributes by playing different types of
mechanistic roles. Among all the post translations modifications, the greater attention is needed
by or given to the Phosphorylation which is due to the fact that the kinases are sometimes more
expressed or muted in disease among which cancer is the most common. Due to this, many
antibodies and inhibitors are developed which also helped in antagonizing the kinases activity
(Alibakhshi and et.al., 2017).
Cell signalling helps in governing the basic cellular activities and coordinating the actions of cell
through the coordination of responses with the cellular microenvironment and the coordination is
also complex. By getting better understanding about the cell signalling, diseases may be treated
in more effective manner and also leads to creation of cures. There are numerous pathways of
cell signalling such as Notch signalling, Akt and NF-kB. The antibodies produced by Rockland
helps in assisting in the research regarding the pathways mentioned. HER2 which is referred to
as human epidermal growth factor receptor 2 is considered as the member of the family receptor
of epidermal growth factor. This HER2 can be dimerized with any other member of the family of
ErbB which helps in auto-phosphorylation of residues of tyrosine and activating the signal
pathways. Then the transfer of signals take place through the different kinase such as STAT,
PI3K/Akt etc. The antibodies of Rockland’s phospho-specific help in detecting the signal. This
also helps in accurately determining the pathway’s activation. This is considered as the
advantage of antibodies as the HER2 does the direct interaction with more than 50 proteins (Yu
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and et.al., 2017). Likewise, the STAT signalling also plays a role in the research related to
immune deficiency along with various categories of cancers. When there is enough knowledge
about the autophosphorylation behaviour of the pathways along with the impact of the STAT
proteins on the other pathways, this can be analysed the interaction of signalling with the
functions of cell. The antibodies help in continuing the research mainly in the field of cancer and
the fields of immunology.
The development of antibodies which are phosphorylation-specific also gives the ability for
measuring the protein phosphorylation. These antibodies are proved to be useful in many
different techniques. The antibodies which are phosphor-specific can be used in multiplexed
bead arrays which are able to be measured and sorted by making use of the Luminex platform.
The antibodies are considered to be immobilized on beads. The amount of the fluorescent dye
identifies these beads. The mixture of beads and the fluorophore-conjugated secondary
antibodies enable the detection of 100+ distinct analytes which are from the same lysate. When
the antibodies are carefully validated along with the protein loadings, Luminex becomes able in
providing the quantitative measurements in which the degree of multiplexing is very high. The
antibodies which are metal-tagged help in labelling the cells which are passed in single file. The
antibodies which are phosphorylation-specific are also used in the high-throughput arrays
(Mayes, Hance and Hoos, 2018).
The limitations of using the antibodies is that sometimes it becomes very expensive and
considerable effort is required for producing them. The culture of hybridoma may be
contaminated. There can be enhanced chances of cross-reaction which are known as false
positives. The antibodies sometimes require multiple numbers of control samples for arriving at
meaningful conclusions. Therefore, special considerations must be considered at the time of
phosphorylation-dependent signalling. The scientific objectives must be decided by considering
the sample, cost and availability of the instruments along with the processing time. These
limitations must be considered practically during the use of antibodies.
2. Using the antibody-based assays to investigate the sub-cellular localisation of a protein.
Antibody-based assays are considered as an attractive option for the diagnostic tests because
these are relatively cheap, highly specific and non-toxic. The immunosorbent assay which are
enzyme-linked are referred as ELISA is basically a test which is completed in few hours and also
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it is performed in 96- or 384- well microplate. This can be also fully automated which also
allows for high sample throughput and the sensitivity is also maintained for analysing accurately.
There are various assays which can be applied commercially and industrially for the locality of
protein (Gao and et.al., 2020). This was observed that when the cells are exposed to radiation,
this changes the activity of cellular proteins. Number of DNA repair enzymes and the number of
paracrine signalling molecules are included like the receptors, cytokines and the adhesion
molecules of downstream cell.
The Luminex technology can be considered as the protein detection methods along with the
sophisticated techniques. This can give the results as the proteins can be found in the blood of the
individuals who are exposed. It can also provide the information regarding the biological
response to the radiation as compared to the profiling of gene expression and also the small
number of proteins can be used to get the accurate dose measurements.
The antibody-based assays also give the results as some proteins induced by irradiation are more
secreted and located and can be displayed on the surface of the cell. The main target for the high-
throughput assays is the antigents as without even lysing the cells, the detection of extracellular
levels and the alterations can be done and the main consideration is the sample preparation ease
regarding the high-throughput assays.
In order to track the extracellular level of protein, the best location is the finger from where
blood can be taken or from cells of the mouth swab which can be assayed quickly.
The control steps which can be taken are extracting the protein which is found in the nucleus.
The quality of antibodies and standards must be maintained along with maintaining the quantity
of the antibodies and standards. The purity of the antibodies and the standards must be
maintained but only when the antibodies which are possible ate affinity purified. The specificity
and the selectivity of antibodies must be done effectively (Robu and et.al., 2020).
This was evaluated that most of the proteins almost more than 260 give effective response to the
radiation. Therefore, various systematic reviews are performed for examining the candidate
antigens. Range of cells must be used which are cultured not only the normal cells but also the
hematopoietic cells which helps in detecting the changes in variety of proteins which are secreted
in which the system used as the detection system is ELISA. This was also examined from the
studies that the expression of the proteins is increased by the cells after the process of irradiation
or not. The another option is that the cells if negative at the initial level can be induced for
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expressing that the protein is above the threshold level regarding the detection in the antibody
based assay. The human cell lines are sometimes selected which are mostly available in the
laboratories and the normal cell lines are chosen instead of the tumour cell lines. Then the
antigens were chosen whose expression of protein changed after expressing it to the radiation. In
most of the proteins, changes were observed. The most important aspect is to develop the
diagnostic assay for the humans along with validating the technique effectively in the complete
organism. The principle tests were done on the organisms mainly by irradiating the mice along
with using the detection test of ELISA for detecting the changes in the levels of cytokine in the
samples of blood (Yin and et.al., 2019). The materials which are found to be analogous which
are acquired by the humans by the prick from the finger. It also has other advantages such as the
mixture in the circulatory system, samples which are obtained from the individuals even if they
have partial exposure of body then also the cells contained will be irradiated directly. This is
how, the proteins are the materials which functions optimally in the particular sub-cellular
localization. Therefore, the main role is played by the effective transportation of the protein to
the final and last designation which affects the overall functioning. Hence, most of the proteins
in the cell in the nuclear DNA in which the small subset is being encoded in the mitochondrial
and chloroplastic DNA. The protein is reached to its last and final destination by the effective
and selective sorting system and the efficient mechanism of transportation. The proteins are also
located in the cytoplasm which can be entering the secretory pathway.
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REFERENCES
Books and Journals
Alibakhshi, A. and et.al., 2017. Targeted cancer therapy through antibody fragments-decorated
nanomedicines. Journal of Controlled Release. 268. pp.323-334.
Gao, Z. and et.al., 2020. Development of antibody-based assays for high throughput discovery
and mechanistic study of antiviral agents against yellow fever virus. Antiviral
Research. 182. p.104907.
Mayes, P.A., Hance, K.W. and Hoos, A., 2018. The promise and challenges of immune agonist
antibody development in cancer. Nature Reviews Drug Discovery. 17(7). pp.509-527.
Robu, M. and et.al., 2020. Methods to Study Intracellular Movement and Localization of the
Nucleotide Excision Repair Proteins at the DNA Lesions in Mammalian Cells. Frontiers
in Cell and Developmental Biology. 8. p.1369.
Yin, R. And et.al., 2019. Localization of SUMO-modified Proteins Using Fluorescent Sumo-
trapping Proteins. JoVE (Journal of Visualized Experiments). (146). p.e59576.
Yu, S. and et.al., 2017. Recent advances of bispecific antibodies in solid tumors. Journal of
Hematology & Oncology. 10(1). p.155.
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