Lab 2.2 Immunoprecipitation
2.2 Immunoprecipitation
Different protocols were compared so as to choose the one which is compatible with the characteristics of the particular protein of interest [29]. A suitable amount of nuclear extracts was diluted with 100 microliters of chilled PBS, 0.1% Triton 100 (to this mixture, the following was added to make a final concentration of 1:1000: 0.2 mM PMSF, 0.5 mM DTT and 15 (microgram/ microliter). It was made sure that the final concentration of nuclear extract was 0.5-1.0(microgram/ microliter). The solution was left for 20 mins at 30˚C (RNase A Treatment). Then it was centrifuged at 15000 RPM for 10 minutes to remove any debris. The supernatant was incubated with an appropriate amount of flag- crosslinked beads at 4˚C for three hours, rotating. Afterwards, the beads-containing solution was centrifuged …show more content…
Preparation and storage of buffers and general solutions used: All Solutions were made with Ultra-pure water (MilliQ water). The 4% formaldehyde solution was freshly made in PBS. (phosphate buffer saline). A 0.1 % solution of Triton X-100 was made prior to use in PBS. The t-RNA solution is made in water at 10 mg/ml and stored in aliquots at −20 °C. The 20× SCC: 175.3 g of NaCl and 88.2 g of sodium citrate are dissolved in 800 ml of water, pH is adjusted to 7 with 10 N NaOH, and the volume is completed to 1 L with water. The Formamide solution, CY3-streptavidine and dextran sulphate are stored at 4 °C. The biotinylated oligo(dT) probe is reconstituted in water at 10μg/μl and stored at −20 °C. A 100 μM stock solution of DAPI is made in PBS and stored at 4 °C in the dark. The Prolong anti-fade kit is stored at −20 °C. Mounting media is made according to the manufacturer’s instructions and is stored at −20 °C. It is advisable to add nitrogen gas over the mounting solution prior to closing the vial. This will prevent oxidation and help store it for a longer
Different protocols were compared so as to choose the one which is compatible with the characteristics of the particular protein of interest [29]. A suitable amount of nuclear extracts was diluted with 100 microliters of chilled PBS, 0.1% Triton 100 (to this mixture, the following was added to make a final concentration of 1:1000: 0.2 mM PMSF, 0.5 mM DTT and 15 (microgram/ microliter). It was made sure that the final concentration of nuclear extract was 0.5-1.0(microgram/ microliter). The solution was left for 20 mins at 30˚C (RNase A Treatment). Then it was centrifuged at 15000 RPM for 10 minutes to remove any debris. The supernatant was incubated with an appropriate amount of flag- crosslinked beads at 4˚C for three hours, rotating. Afterwards, the beads-containing solution was centrifuged …show more content…
Preparation and storage of buffers and general solutions used: All Solutions were made with Ultra-pure water (MilliQ water). The 4% formaldehyde solution was freshly made in PBS. (phosphate buffer saline). A 0.1 % solution of Triton X-100 was made prior to use in PBS. The t-RNA solution is made in water at 10 mg/ml and stored in aliquots at −20 °C. The 20× SCC: 175.3 g of NaCl and 88.2 g of sodium citrate are dissolved in 800 ml of water, pH is adjusted to 7 with 10 N NaOH, and the volume is completed to 1 L with water. The Formamide solution, CY3-streptavidine and dextran sulphate are stored at 4 °C. The biotinylated oligo(dT) probe is reconstituted in water at 10μg/μl and stored at −20 °C. A 100 μM stock solution of DAPI is made in PBS and stored at 4 °C in the dark. The Prolong anti-fade kit is stored at −20 °C. Mounting media is made according to the manufacturer’s instructions and is stored at −20 °C. It is advisable to add nitrogen gas over the mounting solution prior to closing the vial. This will prevent oxidation and help store it for a longer