Cellular Protein Isolation And Analysis

Fractionation of Subcellular protein

In the experiment, nearly 2x106 cells are pelleted and later washed with cold PBS. According to the protocol mentioned by the manufacturers, the Cytoplasmic and Nuclear Extraction Reagents NE-PERTM Kit was used for isolating the nuclear and cytoplasmic protein from the cells. In brief, the kit was seen to allow stepwise preparation and separation of extracts from nucleus and cytoplasm from the cells with 2hours. Moreover, addition of the reagents CER I and II administered to a cell pellet was seen to disrupt the cell membrane and result to release contents of the cytoplasm. After intact nuclei were recovered from extract of cytoplasm by centrifugation method, the proteins were seen to be extracted from the nuclei with the use of NER reagents in another tube which is previously chilled. The BCA assay was used for analysing the concentration of isolated proteins for the purpose of western blot analysis.

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The ECL plus (ThermoFisher Scientific, #32132) is seen to be used in the form of substrate for revealing the western blot signals by following the instructions by the manufacturer that were detected by using various systems such as detection through digital camera with BioradChemiDoc XRS+ imaging system (#1708265) and film exposure (CL-XPosure Film, #34091) that uses optimal exposure times.

Transposable elements present in cancer

The transposons are referred as mobile genetic elements which have the ability to transpose or jump from one site of the DNA to the other. The way of transposition is referred to a form of genetic recombination and it is catalyzed with the assistance of special enzymes know as transposases. In the experiment executed by Barbara McClintock in the maize, the transposition of the genes was first identified (Comfort, 2001; McClintock, 1951). The transposons since then were seen in nature in many instances with them being found in virtually all nature of organisms. There is wide number of sequencing results which have mentioned that a large fragment of DNA present in different eukaryotic genomes are seen to contain transposable elements along with their remnants. For instance, nearly 40% of both the mouse and human genomes are seen to be derived from transposons (Waterston et al., 2002; Lander et al., 2001). The transposition helps in creating variety of rearrangements in the genetic codes like translocation, deletion and insertions.

The transposable elements are seen to be classified into Class I retransposons which use RNA transcription mechanism for copying and pasting themselves and Class II retransposons which is DNA transposons that cut the site of the donor for reintegrating elsewhere through cut and paste mechanisms in the genome (Wicker et al., 2007). The Class II of transposons are seen to comprise 5% of the human genome but they are seen no longer to be in an active state present in the humans. The retransposons contains 45% of the genome present in human and they can be categorised in two types that are long-terminal repeats (LTRs) and non-long terminal repeats (non-LTRs). The non-LTRs are seen to involve interspersed elements (LINE) as well as short interspersed elements (SINE) whereas the LTRs are seen to contain the human endogenous retroviruses (HERVs). The HERVs are seen to be similar in structure for infecting the retroviruses as it is thought that they are acquired from infection of the germline cells by the retroviruses of ancient nature who later through the help of evolution has become a part of the genome. Thus, the genetic sequence mentioned in the HERVs is seen to contain main retroviral genes such as pro, gag, env and pol (Dewannieux & Heidmann, 2013; Stoye, 2012). On analysing the mRNAs as well as the proteins in the active TEs, it is revealed that they are seen in active condition in many cancers (Downey et al., 2015).

The activity of TEs is seen to be regulated by the epigenetic mechanisms. In normal tissues, the genomic sequences present in the TEs are hypermethylated (Schulz et al., 2006). However, in majority of the samples collected from tumour it is seen that the TEs are in a hypomethylated state that is able to promote activity of the genes in the cancers (Shukla et al., 2013; Alves et al., 1996). The piwi-interacting RNA (piRNA) pathway is seen to remain in active state in the germ cells and it is seen to use dicer-independent piRNAs. The piRNAs is seen to be processed by piwi proteins which are able to bind with Ago3 that acts for promotion of cleavage in the antisense mRNAs (Aravin et al., 2007). The piwi proteins are seen to play a similar role in the humans too along with in TE promoter the proteins have the ability to trigger methylation (Moyano & Stefani, 2015). In addition, PIWI family members present in the humans are seen to show aberrant expression in various cancers which may result in dysregulating TEs in the tumours. However, analysing the impact of PIWI proteins it is unclear that whether the normal amount of Piwi protein is per se tumorigenic or it is regarded as a consequence of disease.

EpiQuik Histone H3 Modification Multiplex Assay Kit (Colorimetric)

The Histone H3 Modification Multiplex Assay Kit (Colorimetric) (P-3100-96) is referred to the complete set of reagents in optimised form that are used for detecting and quantifying 21 modified Histone H3 patterns in simultaneous nature in a simple ELISA-like format by using a standard microplate reader. The cancer cells in the humans in the stage of confluence were seen to be seeded in a 6- well plate at the rate of 2×105 cells per well (Costar #3516) by using addition of 10% FBS as a complete medium. After this, the cells were incubated at desired temperature with the presence of desired CO2 percentage overnight for allowing attachment. In the next day, the siRNA treatment was seen to be prepared in the Eppendorf rube which is sterile in nature for each of the wells. The same procedure was executed for the next two days with incubation of the cells for giving them a siRNA treatment. After this, the cells are harvested for extraction of histone proteins. It is estimated that the quantity of purified histone H3 protein for each of the assay is 1ng-25 gm. The extracts of the histone are required to be stored in aliquots at a temperature of -80ºc till it is used by following the procedure mentioned in the manual provided by the manufacturer of the kit.

Analysis of Curve growth

In 6 different well plates, the cells are plated at a certain concentration based on the cell line (105 cells/ml for HCT116 cells and 2x105 cells/ml for SW480 cells) in the well with final volume ranging to 2 ml/well. On daily basis, the treatments of non-interfering RNA/siRNA are applied to the cell as mentioned in the comments before. In the course of the experiment, each of the conditions is carried out in the form of triplicates and the cells are counted after executing trypsinisation by use of trypan blue and TC20 Automated Cell Counter (Biorad, #1450102).

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Table:

  • Image of culture conditions along with the cell lines involved in the study
  • Image of the primers that have been used in the PCR
  • In this experiment, RT-qPCR primers are used
  • The primary antibodies which are used for the Western Blot analysis
  • The secondary antibody concentration used for executing western blot analysis
  • The dilution along with the primary antibodies used for analysing human proteins in the normal tissues by using IF staining
  • Secondary antibodies and the optimal concentration that is used for the immunofluorescent assay
  • Image of the source of normal lysates that are used for the purpose of executing Western Blot analysis

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