In this research paper, the functional relationship between protein named Albert and Alice will be prove using two advance techniques. Most of the molecular processes within the cell are carried out by protein-protein interaction (PPI), diseases such as cancer, Alzheimer’s disease can caused by abnormal PPI. As prior knowledge has advocate that this two proteins may have similar cellular pathway, therefore understanding if they have PPI becomes vital.
There will be two methods described in this paper, Enzyme-Linked immunosorbent assay (ELISA) and proximity ligation assay (PLA). ELISA are reported by Wisdom,G. B. (1976) to be more sensitive than quantitative precipitation, immunofluorescence, agglutination, passive hem agglutination and complement …show more content…
The purpose of this method is to wash away any excess protein that does not bind to the plates.
PLA method
The method is adapted from Söderberg et al’s (2008) research papers. Proximity ligation assay often contains four major part, 1) PLA probes, 2) Ligation, 3) Amplification and 4) Preparation for imaging. The steps are shown below
1. PLA probes: Wash the well with PBT for 10 minutes for 3 times, incubate the PLA probes for 2 hours at 37 degree C.
2. Ligation: Wash the body walls with wash buffer A twice for 5 minutes each, incubate with ligation solution
3. Amplification: Wash the body walls with wash buffer A twice for 2 minutes each, incubate with amplification solution for 2 hours at 37 degree C.
4. Imaging: Wash the well with wash buffer B twice for 10 minutes each, wash with 0.01x wash buffer B once for 1 minute, equilibrate in a few drops of mounting solution for at least 30 minutes before mounting, or store overnight at 4 degree C, using fine forceps, transfer the body cell on a platform. Position the body walls in rows and in the same orientation within a drop of mountant, place coverslip, seal the slide with clear nail polish, and store the slide in the dark at -20 degree C until ready for confocal
There will be two methods described in this paper, Enzyme-Linked immunosorbent assay (ELISA) and proximity ligation assay (PLA). ELISA are reported by Wisdom,G. B. (1976) to be more sensitive than quantitative precipitation, immunofluorescence, agglutination, passive hem agglutination and complement …show more content…
The purpose of this method is to wash away any excess protein that does not bind to the plates.
PLA method
The method is adapted from Söderberg et al’s (2008) research papers. Proximity ligation assay often contains four major part, 1) PLA probes, 2) Ligation, 3) Amplification and 4) Preparation for imaging. The steps are shown below
1. PLA probes: Wash the well with PBT for 10 minutes for 3 times, incubate the PLA probes for 2 hours at 37 degree C.
2. Ligation: Wash the body walls with wash buffer A twice for 5 minutes each, incubate with ligation solution
3. Amplification: Wash the body walls with wash buffer A twice for 2 minutes each, incubate with amplification solution for 2 hours at 37 degree C.
4. Imaging: Wash the well with wash buffer B twice for 10 minutes each, wash with 0.01x wash buffer B once for 1 minute, equilibrate in a few drops of mounting solution for at least 30 minutes before mounting, or store overnight at 4 degree C, using fine forceps, transfer the body cell on a platform. Position the body walls in rows and in the same orientation within a drop of mountant, place coverslip, seal the slide with clear nail polish, and store the slide in the dark at -20 degree C until ready for confocal