Detection Of Ras Raf Protein Interactions Biology Essay

In the recent old ages several methods have been developed to research the protein alterations and interactions as they play an intrinsic function in any cellular and sub-cellular procedures. Several immunohistochemistry techniques were developed for proteome surveies. Proximity ligation check is an affinity based technique used to mensurate protein interaction and alteration at individual molecular degree. Fredriksson et Al in 2002 developed the propinquity dependent DNA ligation check for the in situ analysis of proteins. PLA technique combines the propinquity ligation with rolled circle elaboration that involves the binding of the mark proteins by specific antibodies linked to the oligonucleotides called as PLA investigations. The propinquity probes serve as the templet for the added connection oligos to organize a complete circularized oligo by enzymatic ligation reaction. The circularized DNA strand gets amplified utilizing one of the antibody edge oligonucleotides as the primer to organize RCP ( fig.1 ) . Fluorescence labeled oligonucleotides are hybridized to the RCP, therefore leting to analyze the RCP under fluorescence microscope [ 3, 6 ] .


Fig.1: in situ PLA: Interacting proteins are bound by the antibodies with PLA investigations which so serve as templet for connection oligonucleotides. Ligation followed by RCA occurs to give RCP. The amplified merchandise is farther analysed under fluorescence microscope after crossbreeding with fluorescence investigations.

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Proto-oncogene serine/threonine protein kinase Raf1 maps downstream of Ras protein activation in the Mitogen-activated protein kinase ( MAPK ) signaling pathway modulating the cell-growth, distinction, endurance and programmed cell death ( Fig.2 ) . Ras household includes the proto-oncogenes HRAS, KRAS, NRAS, RRAS. Raf household includes A-Raf, B-Raf and Raf-1 ( Raf-C ) [ 7 ] . Mutants in any of these cistrons leads to over-expression of growing factors finally taking to assorted malignant neoplastic diseases.

Fig.2: Raf activated by Ras protein activates MEK/ERK tract. ( Hilger et al. , 2002 )

In our experiment we used in situ PLA to analyze the Ras-Raf interactions in HCT116 cell line CCL-247 from American Type Culture Collection ( ATCC ) .


The cell slide incorporating the HCT116 cell line CCL-247 ( from ATCC ) was regained from -20 & A ; deg ; C and was placed in 1x tris-base saline ( TBS ) . Immediately the bluish countries were dried off with the aid of napkin without upseting the cell country. Within 15sec, a hydrophobic margin was drawn around the cell countries on the bluish line and the boundary line was dried for 3-4 seconds before puting the slide back into TBS. Then the slide was washed twice with Tween-20 supplemented TBS ( TBST- 0.05 % ) for 2 proceedingss. Before the add-on of any dilutant to the walls in the slide, extra buffer was removed from the slide and it was ensured that hydrophobic margin was integral. Duolink barricading agent ( 2-3 beads ) was added to each well and the slide was incubated for 30 proceedingss at 37 & A ; deg ; C in a humidness chamber followed by a 2×2 min wash in TBST. Each slide has two Wellss in which one will be used for negative control and the other one for unrecorded experiment. Both primary antibodies { Ras ( H- , K- and N-Ras ) from US biological and Raf1 ( C ) from RnD } was diluted in Duolink antibody dilutant to organize a entire volume of 40µl for the unrecorded experiment whereas for negative control, one of the antibodies ( anti-Raf ) was omitted. Each antibody dilution ( 40µl ) was added to the several Wellss and the slide underwent 1hour incubation at 37 & A ; deg ; C followed by a 2×2 min wash in TBST. Duolink II secondary antibodies ( rabbit- and mouse+ ) 5x was diluted in Duolink antibody dilutant to organize a entire volume of 80µl and 40µl of this dilution was added to each well. Then the slide was incubated at 37 & A ; deg ; C for 60min followed by a 2×2 min wash in TBST.

Ligation buffer and ligase was diluted in the ratio of 1:5 and 1:40 severally in H2O to do a entire volume of 80µl and 40µl of this ligation mix was added to each well followed by a 30min incubation at 37 & A ; deg ; C and a 2×2 min wash in TBST. Amplification buffer and polymerase was diluted in the ratio of 1:5 and 1:80 severally in H2O to do a entire volume of 300µl and 150µl of this elaboration mix was added to each well. The slide was so incubated at 37 & A ; deg ; C for 60min followed by a 2×2 min wash in TBST and 2×2 min wash in TBS. Since, the reagents in this measure are light sensitive, the wining stairss were shielded from visible radiation every bit much as possible. The slide was centrifuged for 10sec after the remotion of extra buffer from it followed by an add-on of 25µl Duolink II mounting medium. Immediately, the slide was covered utilizing a screen faux pas and was turn upside- down on a serviette so that extra mounting media was removed. Finally, the coverslip was glued onto the slide by using nail gloss around the borders and was stored from visible radiation in -20 & A ; deg ; C. ( Duolink kit from OLINK BIOSCIENCE, Uppsala ) .


We performed the in situ propinquity ligation check with the cell slide incorporating HCT116 cell line CCL-247 from American Type Culture Collection ( ATCC ) to find the ras/raf interactions. The targeted proteins were bounded utilizing two primary antibodies raised in different species followed by binding of specific secondary antibodies with oligonucleotides called PLA investigations. Upon propinquity the connection oligos gets hybridized to the PLA investigations proceeded by enzymatic ligation and gets amplified by RCA to give RCP. Finally the RCP is analysed by crossbreeding with fluorescence labeled oligos each topographic point stand foring single RCP.

From the figure 3, we can detect the Ras-Raf protein interactions ( highlighted in green coloring material ) in the unrecorded experiment when compared with the negative control. Few green points were besides spotted in the negative control and this may be due to managing mistake. This mistake would hold happened during the remotion of extra buffer from the slide before add-on of any dilutant.

Ras-Raf interactions play a broad function in cell rhythm. In the MAPK tract besides known as Ras/Raf/MEK/ERK, the activated G-protein Ras interacts with Raf1 which in bend phosphorylates the MEK. MEK once more activates ERK by phosphorylating it, of import to command cell growing, distinction and programmed cell death. Mutation of any one of the above proteins leads to over look and activation of growing factors and eventually malignant neoplastic disease. Suppressing the above proteins has been proved to handle malignant neoplastic disease. In Colorectal malignant neoplastic diseases which causes bulk of the malignant neoplastic disease deceases, dysregulation of MAPK tract is thought to be a major cause. Recently several research plants are being carried out to plan the fresh inhibitors for the above potency proteins. These surveies will pave the manner to better intervention to assortment of malignant neoplastic diseases [ 1, 4, 7 ] .