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In situ Proximity Ligation Assay (PLA) is used to study protein-protein interaction in the cells. This technique is also used to identify the new drug multifaceted molecule with the help of binding assay from the chemical compound library. Post translational modifications (PTM's) are common events in eukaryotic cells, any modifications leads to aberrant signalling. This method makes use to of two separate modified antibodies attachment with oligonucleotides to target with two recognition region in protein complex. Up on proximal binding the oligonucleotides on PLA probes are brought together and ligated by enzymatic fusion to form circle DNA. This circle DNA is amplified by rolling circle amplification (RCA) on one of the attached antibody and the amplified DNA is detected by hybridization of fluorescently labelled probe to the repetitive sequence. In this experiment we analysed the β-catenin - or / E-Cadherin interactions in CLL-247 cells by Duolink PLA kit. In live experiment there was signal indicating the β-catenin/ E-cadherin protein interactions. But in the case of negative control the fewer signals were due to the drying of cell surfaces during subsequent washing steps.
In situ Proximity Ligation Assay (PLA) was first described by Ola Söderberg et al.,  in 2006. This principle is mainly used to study protein-protein interaction in the cells which regulates the cell signalling that mainly controls the cellular mechanisms like migration, proliferation and apoptosis . This assay is also used for screening the new drug multifaceted with the help of binding assay to identify molecule from the chemical compound library. Then the selected chemical molecules are used for supplementary branded in the derivative from cell-related assays to remove the unwanted unrelated molecule .
Post translational modification (PTM) is normally occurring events in eukaryotic cells. PTM's are the innumerable, common most are phosphorylation. These modification leads to downward signalling from the cell surface to the cytoplasm and end in the nucleus. Commonly PTM's are formed by the enzyme mediated impulsively in case of phosphorylation and glycosylation . These PTM's are studied by immunofluorescence methods, as it is identified by the antibody that binds to protein complex by the fluorescent labelled probes. This method is simple and efficient in identifying protein interactions by antibody based recognition .
In situ PLA is very extremely sensitive and discriminatory methods used for the detection of protein activity, PTM of protein and protein-protein interactions. It is also used in many application of protein based detection. This method makes use to target of two recognition region of protein complex by the help of the two separate modified antibodies attachment with oligonucleotides. The two antibodies bind to an epitope of the protein complex. On proximal binding the oligonucleotides on PLA probes are brought together and ligated by enzymatic fusion the form of circle DNA. The circle DNA amplified by rolling circle amplification (RCA) in the form of antibody attached to the oligonucleotide act as a primer. The rolling circle amplification is a long length of DNA formed by single stranded chain by the circle replication. This can be detected by the hybridization of fluorescently labelled oligonucleotide to the repetitive sequences. The targeted cell are display by the high florescent easily detected by the help of the florescence microscope and measure by the imaging analysis .
Materials and method
Cell slide preparation and Blocking
RKO cell line from American Type Culture Collection (ATCC) was cultured in 2 wells on cell slide. One well was marked as live and other as negative. Initially cells were washed in 1x tris-base saline (TBS) and hydrophobic perimeter was immediately drawn around cells. Later the slide was washed twice in Tween supplemented TBS (TBST) (0.05%). After washing excess buffer was flick off and making sure that hydrophobic perimeter was intact. Duolink blocking agent of 3 drops was added to each well and incubated at 37°C for 30 minutes in homemade humidity chamber. Finally slide was washed twice in TBST.
For live well, both primary antibodies (β-catenin [1:80] / E-Cadherin [1:10]) (Duolink kit) was diluted to total volume of 40 µl using Duolink antibody diluent and for negative well only (E-Cadherin [1:10]) was diluted to 40 µl using Duolink antibody diluent was prepared in separate tubes. The prepared solution was respectively added to appropriately labelled wells and incubated at 37°C for 60 minutes in homemade humidity chamber. After incubation slide was washed twice in TBST. Secondary antibodies [1x] (rabbit- and mouse+) (Duolink kit) was prepared using Duolink antibody diluent. By flicking off excess buffer from the cell slide, 40 µl of diluted secondary antibodies was added to each well and see to that hydrophobic perimeter was intact. The cell slide was incubated at 37°C for 60 minutes in homemade humidity chamber. After incubation slide was washed twice in TBST.
Ligation and Amplification
Ligation buffer and ligase (Duolink kit) was diluted to 1:5 and 1:40 respectively to total volume of 80 µl with water. By removing the excess buffer from the slide, 40 µl of ligation mix was added to each well and incubated at 37°C for 30 minutes in homemade humidity chamber. After incubation slide was washed twice in TBST. For amplification, amplification buffer and polymerase (Duolink kit) was diluted to 1:5 and 1:80 respectively to total volume of 300 µl with water. Excess buffer was removed and 150 µl of amplification mix was added to well and incubated at 37°C for 60 minutes in homemade humidity chamber. After incubation slide was washed twice in TBST following by TBS.
Mounting and imaging
After final wash excess buffer was removed and slide was briefly centrifuged. Duolink mounting medium (Duolink kit) of 25 µl was added on both the wells and covered with cover slip. Using napkin excess of mounting medium was removed and glued by using nail polish around the edges of the slip. The imaging of slide was taken by fluorescent microscope.
Results and Discussion
Here we analysed the β-catenin - or /E-Cadherin interactions in CLL-247 cells by In situ-PLA approach. This technology is based on visualising an interaction between two target molecules (Figure 2A). First, the primary antibodies are directed to proteins of interest, following by secondary antibodies conjugated to connector oligonucleotides are incubated (Figure 2a). Second, is the hybridization of connector oligonucleotides if the proteins are in close proximity (Figure 2b).Third, is the enzymatic ligation of PLA oligonucleotides (Figure 2c). Amplication step to form rolling-circle amplification reaction (RCA) products of long single-stranded DNA molecule (Figure 2d). Lastly, the hybridization of RCA product with fluorescently labelled oligonucleotides (Figure 2e). Thus the fluorescent signals generating from RCP products enables in vivo the protein - protein interaction.
Beta-catenin (β-catenin) is a transcription factor encoded by the CTNNB1 gene in humans. It has been known as an integral component in the Wnt signaling pathway. β-catenin plays as an oncogene and it has been shown that increase in β-catenin production several carcinomas and colorectal and ovarian cancer. This protein is mainly involved in Wnt signalling pathway . E-cadherin is a 97-kDa transmembrane glycoprotein encoded by the E-cadherin gene (CDH1). They mainly acts as adhesion protein of the epithelia thus ensuring that cells are bound together and also functions as calcium-dependent cell attachment and the development of tissue architecture. The binding property of β -catenin, is stabilized by E-cadherin. This complex maintains the epithelial cell adhesion and architecture . Any mutation or disruption in this protein complex leads to in tumor progression including benign melanocytic, malignant melanomas and gastric carcinoma [10,11].
C:\Users\Venkatesh\Desktop\Lab reports\PLA\exp.jpg C:\Users\Venkatesh\Desktop\Lab reports\PLA\neg ctrl.jpg
Figure 1 - In situ PLA of β-catenin/ E-cadherin. A) Schematic principle of In situ PLA. Different steps in the protocol corresponds to (a-e). This image is taken from Vuoriluoto et al, (2011) . B) Live experiment of CLL-247 cells grown glass slide and treated with both primary antibodies of β-catenin/ E-cadherin. After fixation, corresponding secondary antibodies containing PLA oligonucleotides are added and amplified followed by hybridization with fluorescently labelled oligonucleotides. The detected dimers (β-catenin/ E-Cadherin) are represented by the fluorescent rolling circle products (red dots) and nuclei are represented by blue colour. C) Negative control is treated with only E-Cadherin primary antibody and remaining steps were followed similarly as live experiment.
The negative control was performed in order to avoid the background signals. The rolling circle amplification (RCA) products are produced only when the two antibodies are in close proximity as in the case of live experiment, whereas in negative control which is devoid of a primary antibody. As this method uses dual target recognition of the protein complex by a pair of antibodies and hence RCA products are not produced by only one primary antibody, since the two secondary antibodies cannot bind in close proximity.
Here, in live experiment the red dots indicates the β-catenin/ E-cadherin protein interactions in vivo and blue dots represents the cell nuclei. But in the case of negative control there were few signals. These signals are due to the drying of cell surfaces during subsequent washing steps. If the cells get dried up, then the antibodies struck in the medium leading to nonspecific hybridizations of fluorescently labelled oligonucleotides and thus generating random signals.