This procedure begins with culturing the bait strain in media containing normal amino acids in the SLAC control strain and heavy isotopic lysine and arginine. Equal amounts of both strains are mixed and lysed to by grinding and liquid nitrogen Crude lysates, first pelleted by centrifuge and then cleared by binding to spheros beads. Cell lysate is then bound to IgG beads.
The proteins bound to IgG beads are alluded by protease, cleavage and precipitated. The sample is now ready for analysis by mass spectrometry. Hi, I'm Jesse Chao from the Lone Lab in the Department of Cell Environmental Biology at the Life Sciences Institute of the University of British Columbia.
Today show you a procedure for quantitative proteomic analysis called silak. Combined with affinity purification. We use this procedure in our laboratory to study protein complexes.
So let's get started In this procedure, the bait strain contains normal amino acids while the SLAC control strain contains heavy isotopic, lysine, and arginine. To get started, first, grow one liter, each of the bait strain and the control strain separately to the value shown on your screen. Next, pour the culture into 500 milliliter Nalgene centrifuge tubes.
Balance the load in pellet cells at 2, 580 RCF decant the media and resuspend the pellet. With 50 milliliters of cold D H2O while transferring the cells to 50 milliliters, centrifuge tubes, centrifuge the tubes to pellet the cells at 3000 RCF. At this point, you can freeze the pellet at negative 80 degrees Celsius.
It is important to remember to match the control strain culture to the bait strain culture in one-to-one by dry pellet weight. Resuspend each cell pellet in 10 milliliters of NP 40 buffer with a one to 2000 protease inhibitor cocktail. Mix the two cell cultures by directly decanting one tube to the other.
To begin grinding the cells first, pour liquid nitrogen into the pre chilled mortar and allow it to completely evaporate. Next, add two milliliters of cells to the mortar. Pour some liquid nitrogen to freeze the cells.
Grind the cells until the cells become fine.Powder. Repeat the process until all the cells are ground. Be sure to not allow the cells to thaw.
Add liquid nitrogen when necessary. Next, transfer the powder to an ice cold beaker and thaw room temperature. When the edges begin to thaw, add 20 milliliters of NP 40 buffer with a one to 200 PIC after it has thawed.
Transfer the crude lysate to 40 milliliter Nalgene tubes and spin at 16, 000 RPM in a Sovos SA 600 rotor for 30 minutes. While waiting, take 300 microliters of the SROs two B beads and wash the beads in 300 microliters of NP 40 buffer three times. Next, resuspend the beads in 300 microliters buffers so that they are in a one-to-one slurry.
The next step is to pre-clear the cell lysate. To do this first, transfer the super natin to a new tube and add the Spheros two B beads. Incubate on a rotating platform in a cold room for 30 minutes.
While incubating, take 300 microliters of IgG SROs six beads and wash the beads in 300 microliters of NP 40 buffer three times. Then resuspend the beads in 300 microliter buffers so that they are in a one-to-one slurry. After incubation, pellet the SROs.
Two be beads in. Transfer the supernatant to a new tube. Remember to save an aliquot of the cell lysate for western blotting later.
Then add the IgG beads. Separate the contents into two tubes for more efficient binding, incubate on a rotating platform in a cold room for two hours. After incubation, collect the beads in a chromatography column at four degrees Celsius.
Be sure to save an aliquot of the unbound faction. Wash the beads with 10 milliliters. NP 40 buffer.
Next, wash the beads with three milliliters. TEVC buffer. Again, remember to save an aliquot of the beads.
This will reflect the efficiency of binding to elute the beads. First, add five microliters of A-C-T-E-V to one milliliter of TEVC buffer. Add TEVC buffer to the beads and mix.
Transfer the contents into two 1.5 milliliter EINOR tubes from more efficient mixing and incubate on a rotating platform in a cold room overnight. Next, spin down the beads and transfer the eluate to a fresh 1.5 milliliter tube. Wash the beads with additional 0.5 milliliters of TEVC buffer after washing.
Combine the eight in one tube. You should have 1.5 milliliters of ELU eight in total. Save an aliquot of the TEV Elu eight to precipitate.
Adjust aliquots to 25%TCA with a hundred percent TCA. To do this, separate the 1.5 milliliter LU eight into two tubes of 750 microliters each. Add 250 microliters of 100%TCA to the tube.
Your solution should turn milky. Next, place it on ice for 30 minutes. Periodically vortexing the mixture after incubation.
Spin at maximum speed in the tabletop centrifuge in a cold room for 30 minutes. Wash once with 500 microliters of ice cold acetone containing 0.05 normal hydrochloric acid. Then spin for five minutes at maximum speed at four degrees Celsius.
Next, wash once with 500 microliters of ice cold, acetone and spin for five minutes at a maximum speed at four degrees Celsius. After spinning, carefully remove the acetone and dry pellet for silver staining. Resuspend the pellet in 50 microliters of one times the SDS sample buffer.
If you are planning to use the precipitated protein for mass spectrometry based analysis, then it is recommended to precipitate using ethanol. To do this first, add 20 micrograms of glycogen next dilute samples five times with a hundred percent ethanol and adjust to 50 millimolar sodium bicarbonate with a 2.5 molar stock at pH 5.0. Mix the solution by inverting the tube.
Let it stand for two hours at room temperature. Finally, split the contents into two einor tubes. Pellet the precipitated protein by centrifugation for 10 minutes at 12, 000 GS at room temperature.
The aliquot saved during the purification procedure should include the pre-cleared cell lysate, the bound fraction, the unbound fraction, and the eluded fraction. We recommend analyzing the protein contents of the above aliquots by Western blotting using Antit TAP antibody or silver staining to reflect the binding and eluting efficiency of the experiment. In this silver stain gel purified SCS two tap at its interacting, partners were visualized and compared to an untagged control in this Western blot.
Various factions taken during the purification of SCS two tap were analyzed by Western blotting using Antit tap antibody. Notice that SCS two tap migrated at a lower molecular weight after elucian by TEV protease cleavage, and there was no S SCS two tap left on the IgG beads after elution. This confirmed that the purification was highly efficient.
We've just shown you how to purify protein complexes by affinity purification with IgG beads. When doing this procedure, it's important to remember to be sure to match the base strain and the control strain by dry pelle weight before you begin the purification procedure. Since LAC provides us with unbias and qued measurements of protein binding partners, we only carry out the first stage of the tap purification to minimize sample loss.
If you experience significant amounts of unspecific binding, you may wish to carry out the entire protocol, which can be found in the cold springing harbor manual. So that's it. Thanks for watching and good luck with your experiments.
Hey, come on, mark. Ah, go.
