Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

The overall goal of this procedure is to detect simulation and ubiquitination of budding yeast kinetic core proteins, NDC 10 and NDC 80. This is accomplished by first harvesting yeast cells that express hiss flag tagged SMT three and MT tagged NDC 10 or NDC 80, and making protein extracts from these cells. The second step is to purify hiss flag tagged SMT three conjugates for the detection of summation or to immuno precipitate mik tagg kinetic core proteins for the detection of ubiquitination.

Subsequently, sum ation or ubiquitination is detected by western blot analysis. Ultimately, the presence of a laddering pattern for NDC 10 and NDC 80 confirms that these proteins are simulated and ubiquitinated. The protein purification method described allows the detection of both simulation and ubiquitination of the connico proteins ND C 10 and NDC 80 in the budding yeast sacro.

My cci. The main advantage of this technique is the strain we have created has two epitope attacks, one on the test protein, and the other that allows detection of simulation. The use of these tags reduces the background due to cross reactivity that is frequently observed when polyclonal serum are used.

The demonstration of this procedure will be done by Canero Uni, a research fellow in my laboratory. Proteins of interest will be extracted from yeast cell pellets prepared previously and stored at minus 20 degrees Celsius. Resus, suspend the cells in 0.5 milliliters of ice cold buffer.

Use guine buffer if the extract is for a pull-down assay using nickel nitrile, tri acetic acid or nickel NTA superflow beads. Use buffer A if the extract is for immunoprecipitation. Keep the tubes on ice at all times.

Transfer the cell suspensions to two milliliters. Screw cap twos to each tube. Add a volume of glass beads equal to the volume of the cell suspension bead.

Beat the cells in a mini bead beater for two minutes at room temperature and then place the tubes on ice for two to three minutes. Repeat the bead beating and icing three times. Vortex on high speed at four degrees Celsius for 30 to 60 minutes.

Check the cells by visualization under the microscope to ensure that the cells are lysed. Lysed cells will appear as dark ghosts and lack a boundary or defined shape. Optimally at least 80%of the cells should be lyed.

Next, use a push pin to puncture a hole in the bottom of the tube. Place the screw cap on loosely and place the tube in a 15 milliliter conical tube. Centrifuge at 1000 times G for one minute.

To collect the lysate, transfer the lysate to a micro centrifuge tube centrifuge at 15, 000 times G for 30 minutes at four degrees Celsius to collect the extracted proteins. After measuring the concentration of the extracted proteins using a protein assay kit, normalize all extracts so each would contain the same amount of protein. Bring the total volume to one milliliter with the appropriate buffer.

Approximately five milligrams of total protein is obtained from 50 OD 600 of cells. Save 50 microliters of the extracted protein as the whole cell extract. The remaining 950 microliters of protein extract will be used later for protein purification or immunoprecipitation of MT tagged proteins.

Add 50 microliters of two x laly sample buffer to the 50 microliters of whole cell extract. Incubate the samples at 100 degrees Celsius in a heat block for three to five minutes before analyzing them by western blot. Begin this procedure by preparing the nickel NTA superflow beads necessary for the purification.

Obtain the amount of beads needed by low speed centrifugation for one minute. Remove the supernatant and wash the beads with PBS. Add one milliliter of PBS and invert the tube top over bottom until the beads are resuspended.

Collect beads by low speed centrifugation and remove the supernatant. Wash the beads in this manner a total of five times. Suspend the beads in one milliliter of guine buffer and aliquot them into the number of tubes corresponding to the samples to be processed.

Collect the beads by low speed centrifugation and remove the supernatant for each sample. Add 950 microliters of the whole cell extract. Prepared earlier to 100 microliters of the beads.

Incubate on a rocking platform at four degrees Celsius for at least four hours or overnight. Centrifuge at 800 to 1500 times G for one minute at four degrees Celsius. Save 50 microliters of the supernatant.

Add 50 microliters of two x laly sample buffer to this supernatant and incubate at 100 degrees Celsius in a heat block for three to five minutes. 10 microliters of each sample will later be analyzed by western blot. Wash the nickel NTA superflow beads once with one milliliter of guine buffer for five to 10 minutes on a rocking platform at room temperature.

Next, wash the beads five times with one milliliter of breaking buffer after the final wash with breaking buffer resuspend the beads in 90 microliters of two x laly sample buffer. Add 10 microliters of one molar iole to help dissociate the his tagged protein from the nickel NT.A beads incubate at 100 degrees Celsius in a heat block for three to five minutes. Vortex then centrifuge at 13, 000 times G for 30 seconds.

Transfer the supernatant to a fresh tube. To begin this procedure, obtain the necessary amount of anti cmic aero affinity gel antibody by low speed centrifugation. Remove the supernatant, wash the resin with buffer A Add one milliliter of buffer A and invert top over bottom until the resin is resuspended.

Collect the resin by low speed centrifugation and remove the supernatant. Wash a total of five times. Suspend the resin in one milliliter of buffer A and aliquot into the number of tubes corresponding to the samples to be processed.

Collect the resin by low speed centrifugation and remove the supernatant. Add 950 microliters of whole cell extract to 25 microliters of resin Incubate on a rocking platform at four degrees Celsius overnight on the following day. Centrifuge at 800 to 1500 times G for one minute at four degrees Celsius.

Save 50 microliters of the supernatant. Add 50 microliters of two x lam ly sample buffer to this supernatant and incubate at 100 degrees Celsius in a heat block for three to five minutes. Wash the resin with buffer a add one milliliter of buffer, a invert top over bottom until the resin is resuspended.

Collect resin by low speed centrifugation and remove the supernatant after removing the supernatant from the final wash. Resuspend the resin in 100 microliters of sume sample buffer incubate at 100 degrees Celsius in a heat block for three to five minutes. Vortex then centrifuge at 13, 000 times G for 30 seconds.

Transfer the supernatant to a fresh tube. Lastly, load all the samples collected from this protocol. In an SDS page, gel for Western blot analysis, yeast strains that express poly histamine flag tagged SMT three or HF SMT three and MT tagged NDC 80 and NDC 10 were used to detect simulation of kinetico proteins.

HF SMT three substrates were affinity purified using nickel beads and detected with an Anti-Flag antibody. Sum moated NDC 80 was detected in the HF SMT three substrates when probed with an anti mic antibody. The absence of modified proteins in the control strains without HF SMT three indicates the specificity of interaction between HF SMT three and its target proteins.

Moreover, summation of NDC 80 and NDC 10 is reduced in NOCO desole treated cells to detect ubiquitination of NDC 80 and NDC 10. Mick tagged ND C 80 or ND C 10 was immuno precipitated and western blot analysis was performed with anti mic and anti ubiquitin antibodies analysis of whole cell extract and supernatant confirmed the expression of NDC 80 M and NDC 10 M immuno precipitated samples probed with anti MIC showed multiple high molecular weight bands suggesting that NDC 80 and NDC 10 contain post-translational modifications. A laddering pattern of immuno precipitated samples probed with anti ubiquitin showed that ND C 80 and ND C 10 are ubiquitinated.

The laddering patterns of NDC 80 and NDC 10 were enhanced by treatment with a proteasome inhibitor MG 1 32. Further confirming that these represent poly ubiquitination After watching this video, you should have a good understanding of the protein purification technique that allows detection of mation and vaccination of bat NN.