Hi, I'm Stephanie Resi from the laboratory of Lauren Parce in the Department of Molecular Microbiology and Immunology at Brown University. Today we will show you a procedure for the generation of K LRG one Tetramers. We use this procedure in our laboratory to study K LRG one coherent interactions.
Let's get started. First, inoculate four 500 milliliter flasks of TB buffer containing appropriate antibiotics with bacteria expressing the K LRG one plasmid construct and grow at 37 degrees Celsius with shaking when the OD reaches 0.6, angstroms at 600 nanometers induce expression via the T seven promoter with the addition of 0.4 molar IPTG and incubate the culture for an additional four hours shaking at 37 degrees Celsius. Following the four hour incubation, transfer the bacteria to centrifuge tubes and spin the cultures down for 20 minutes at 3000 RPM and four degrees Celsius.
If necessary, pellets can be frozen at minus 80 degrees Celsius at this point for use at a later time. Resus suspend the harvested cells in 30 milliliters of Resus suspension buffer Next pool. Bacterial Resus suspensions up to a 60 milliliter volume in a polypropylene beaker.
If necessary, bring the total volume up to 60 milliliters with extra te buffer PH eight and stir the mixture at half speed on a hot plate stir while the pooled Resus suspension is stirring at the following solutions. Dropwise to the mixture Lysozyme magnesium chloride D NS one TRITTON X 100. And finally DTT.
Transfer the solution to ultracentrifuge tubes using a fisure dismember with ultrasonic converter at 30%amplitude. Sonicate the solution on ice for 1.5 minutes and centrifuge the lysate at 5, 000 RPMs for 10 minutes at four degrees Celsius, decant the supernatant and add 15 milliliters of wash buffer. Repeat sonication as before until the pellet is completely resuspended and repeat centrifugation, repeat the washing fornicating and pelleting steps five times.
Finally, resus suspend the pellet using wash buffer that does not contain Triton X 100. Spin down once more and Resus suspend in four milliliters of TE buffer. These are your inclusion bodies.
Quantify your inclusion bodies by wet weight. The inclusion body slurry can be kept at four degrees Celsius in te buffer stocks of 30 to 100 milligram per milliliter concentrations. With the inclusion bodies ready, you can now prepare for refolding.
Begin the refolding procedure by preparing a fresh one liter bottle of Refolding buffer and stir at low speed at four degree Celsius to chill. Next, you'll prepare seven injections of the inclusion bodies for injection into the refolding buffer. First, melt the stock of inclusion body slurry in a micro centrifuge tube containing a final volume of 1.4 milliliters in seven molar guine hydrochloride plus 10 millimolar beam mercaptoethanol in a water bath at 37 degrees Celsius for 30 to 40 minutes.
Vortexing every five minutes. You will know the melting is complete. When the slurry becomes clear.
Centrifuge the melted mixture at max speed for 30 minutes in a micro centrifuge at four degrees Celsius. Then being careful not to collect any of the small blackish pellet. Transfer the supernatant to a 15 milliliter centrifuge tube.
Adjust the volume to seven milliliters with injection buffer to inject. First, increase the stirring speed of the folding mixture to high. Then add one milliliter of diluted inclusion bodies.
Drop by drop with two seconds between each drop only. Add one milliliter of diluted inclusion bodies at a time with an hour in between each injection in between injections. Return stir speed to low o.
Continue stirring at low speed at four degrees Celsius overnight. The proteins will then be ready for concentration and purification. To begin concentrating the refolded KLRG one tetramers.
First pre-filter the reaction using a point 22 micrometer filter. Then concentrate the reaction from one liter to about 10 milliliters using a millipore Amon stirred cell and a 10 kilodalton nominal molecular weight limit ultra filtration membrane according to manufacturers and S instructions. Then using a Millipore 10 kilodalton molecular weight cutoff ultra filtration membrane concentrate further to less than or equal to two milliliters.
Have your chromatography system set up according to manufacturer's instructions. We use the GE healthcare ACTA fast protein liquid chromatography system at four degrees Celsius using a high resolution column. Purify the monomer form of K lrg, one in 20 millimolar heaps and 150 millimolar sodium chloride by size exclusion chromatography.
The volume injected should be less than 2%of the total gel volume of the column. Again, using a Millipore 10 kilodalton molecular weight cutoff ultra filtration membrane concentrate further to a final volume of one milliliter ate the KLG one monomer according to manufacturer's instructions. Eliminate the free biotin by purifying as before using size exclusion chromatography to achieve tetramer.
Mix the KL LRG one monomers with a fourfold molar excess of FICO rine conjugated streptavidin tetramers are now available for labeling receptor ligands. This graph shows a positive result of separation of Refolded K lrg one proteins using size exclusion chromatography from left to right. The peaks illustrate the multimer dimer and monomer forms of K LRG one.
The peak on the far right represents the buffer exchange. Though refolding conditions have to be tested empirically for each protein, other C type lectin receptors should be able to be tetramer using a similar protocol, thus enabling the potential identification of other ligands. We've just shown you how to generate the KLRG one teer when doing this procedure.
It's important to remember to check the induction levels and to strictly adhere to the refolding conditions. So that's it. Thanks for watching and good luck with your experiments.
