The aim of this procedure is to purify recombinant his tagged proteins in a high throughput manner using an automated protocol on a robotic platform. This is accomplished by first over expressing the proteins in e coli using small volume cultures. Next, the cells are chemically lyed to release the proteins that then bind to magnetic nickel beads.
Then the beads are washed to remove contaminating proteins and to enhance the purity of the desired proteins. Finally, the proteins are purified from the magnetic beads. Ultimately, results can be obtained that show the concentration and purity of the proteins through BCA assays and SDS page and additional functional assays to determine the activity of the proteins.
The main advantage of this technique is that we can now purify a large number of proteins under identical conditions with minimum intervention. This allows us to obtain a high degree of reproducibility and it also minimizes human error. Now this method is particularly useful to study large numbers of point mutants of a particular protein, as it ensures that variability in the activity of the protein is actually due to the mutation and not the differences in the preparation of the samples.
To prepare overnight bacterial cultures begin by filling 24 well plates with water and microwaving them for two minutes to sterilize them afterwards. Discard the water and microwave the plates again for two minutes using freshly grown lack inducible, BL 21 Rosetta two, bacterial colonies or frozen glycerol stocks, inoculate 1.5 milliliters of auto induction medium containing a mixture of glucose and lactose reserve. Three wells each for positive and negative controls, and one well from medium only.Control.
Grow the cells at 250 RPM for 18 hours at 37 degrees Celsius. Remove the lid and place the plate on the robotic platform. The platform contains a gripper arm to move objects from one position to another, a pipetting arm and various positions for the plates and troughs, including a shaking platform, a magnetic bead stir, and a magnetic platform.
For 96 well plates prepare the wash and dilution buffers for the purification procedure according to the written protocol, using 100 milliliters of distilled water and 15 microliters of an anti foam reagent. Prepare a solution for diluting overnight cultures for optical density measurements. Make a lysis solution containing fast break reagent lyase and magnesium acetate and prepare BCA protein assay solution.
As TF two B tends to stick to the pipetting needles, prepare a six Molo Guine hydrochloride solution for washing them. Fill troughs or plates of the right size for the appropriate buffers and place them in their pre allocated positions on the robotic platform, on their positions on the platform. Place 1 24 well plate for the guanine hydrochloride waste two clear 96 well plates for the OD 600 and absorbance measurements, and one blue 96 well plate for the purified proteins.
Place 1 96 deep well plate on the magnetic stand. Use water to dilute magna hair nickel particles and add them to the bead staring unit. Turn on the stir to keep the beads in suspension.
Switch on the robotic platform and flush the pipetting needles for several minutes. To remove air bubbles, place the plate on a shaking platform to check the growth of overnight cell cultures. Dilute 10 microliters of culture in 90 microliters of diluent solution while washing the pipettes between additions.
Then measure the density at OD 600 when the cultures have reached similar densities. Transfer 100 microliters of magnetic nickel bead suspension into the wells of a 24 Well plate transfer 900 microliters of each culture into the wells washing the pipettes between steps. Then add 100 microliters of fast break Lyase mix.
Place the plate on the shaking platform at 800 RPM for 30 minutes at room temperature to allow the cells to lyze and for the his tagged proteins to bind to the beads to wash the beads. Transfer them to a 96 well plate on a stand with magnetic rods that draw the beads away from the centers of the wells, discard the supinate and use six mo iGuide hydrochloride to wash the pipette tips. Next, add 500 microliters of wash buffer to the wells of the 24.
Well plate and transfer the samples to the 96 well plate. Then remove the supinate. Add 500 microliters of wash buffer to the 96.
Well plate. Transfer the plate to the shaker and shake the plate vigorously for one minute. Then move the plate back to the magnetic stand, and remove the wash buffer.
After the final wash, add 100 microliters of elution buffer to the beads and shake vigorously for 30 minutes at room temperature. Then transfer the eluate to a new plate. To quantify the protein samples, add 190 microliters of BCA reagent to the wells of a clear 96 well plate and add 10 microliters of the protein samples to the wells.
Incubate the samples for at least five hours and measure the absorbance with a set of BSA standards. The proteins are then stored at minus 80 degrees Celsius. The purification protocol offers two quality control stages.
First at the bacterial growth stage, and later when assessing protein yields. By purifying the proteins in triplicate, it can be determined that any variation in the functional assays are due to the mutant phenotypes and not caused by experimental variations or failed purifications Yields typically range from 50 to 200 micrograms and are more than sufficient for functional assays. While once master, the technique only takes around three hours, and apart from setting up the platform, setting up the cultures, and then collecting the purified proteins, there's nothing else you have to do.
Following this procedure, functional assays can be performed. In our case, these would be a range of transcription assays in order to investigate how the mutation of TF two B alters its effect on the catalytic activity of ar polymerase.
