It has been well established that the structures of many proteins are stabilized in covalent disulfide linkages. In recent work this bond has been classified as a post-translational modification. Thus, it's important to be able to identify cysteine-stabilized multimer complexes in living cells using a method that's quick and simple.
The main advantage of this technique is that the results can be obtained and interpreted quickly, easily and with minimum cost. To begin, grow U-2 OS cells in a six square-centimeter dish in Minimum Essential Medium High Glucose, supplemented with 10%FBS and 1%sodium pyruvate at 37 degrees Celsius in 5%carbon dioxide. Make a fresh stock of 10 millimolar iodoacetamide just prior to use.
After cells reach 50 to 60%confluency, add 0.1 millimolar final concentration iodoacetamide directly to the cell culture media. Gently rock the dish at room temperature for two minutes. Next, aspirate the media from the cells, and wash the cells with five milliliters of cold PBS three times.
Aspirate the final PBS wash solution, and add one milliliters of cold PBS. Scrape the cells off the bottom of the dish using a cell scraper. Using a one-milliliter pipette draw up the PBS and cell suspension and dispense all the liquid into a 1.5-milliliter microcentrifuge tube.
Spin the cells at 7, 500 times g in four degrees Celsius for three minutes. Aspirate the PBS leaving the cell pellet behind. The cell pellet can be stored at minus 80 degrees Celsius until processing.
To extract the protein, prepare 100 microliters of a 1X lysis buffer diluted in double distilled water. Add PMSF immediately before use to a one millimolar final concentration. Then add 50 microliters of the 1X lysis buffer directly to the cell pellet and re-suspend.
Incubate the extract on ice for five minutes. Sonicate for eight seconds using the constant pulse mode at 40%keeping the extract on ice. Spin the extract at four degrees Celsius and 16, 000 times g for five minutes.
Perform Bradford analysis to determine the protein concentration if desired. To prepare the sample take 10 microliters of the soluble protein extract and add 10 microliters of a 1X Laemmli SDS sample buffer. Keep the samples on ice.
Heat the sample at 85 degrees Celsius for five minutes before running the gel. To begin the formaldehyde cross-linking, grow U-2 OS cells in a 175 square centimeter flask to 70 to 80%confluency. In a fume hood add the formaldehyde fixative directly to the medium to a final concentration of 1%and incubate at room temperature with gentle agitation for 15 minutes.
To quench the reaction add 1.25 molar glycine to a final concentration of 0.125 molar and incubate at room temperature with gentle agitation on a rocker for five minutes. Wash the cells with five milliliters of cold PBS three times. Aspirate the final PBS wash solution and add 10 milliliters of PBS.
Scrape the cells off the bottom of the flask using a cell scraper. Using a 10-milliliter pipette draw up the PBS and cell suspension and dispense all of the liquid into a 15-milliliter conical centrifuge tube. Spin the cells at 500 times g and four degrees Celsius for two minutes.
Aspirate the PBS leaving the cell pellet behind. Prepare 10 milliliters of the homogenization buffer by adding 0.25 molar sucrose, one millimolar EDTA, 10 millimolar HEPES and 0.5%BSA at pH 7.4. Immediately before use, add PMSF to a one millimolar final concentration and three milliliters of the nuclei suspension buffer.
Add five milliliters of the homogenization buffer directly to the cell pellet and resuspend completely. Centrifuge the suspension at 500 times g and four degrees Celsius for two minutes. After centrifugation, discard the supernatant and resuspend the pellet in five milliliters of the homogenization buffer.
Then with a tight fitting glass Teflon homogenizer dounce homogenize the cells at 10 strokes per 500 RPM and centrifuge the suspension at 1, 500 times g and four degrees Celsius for 10 minutes. After centrifugation discard the supernatant. Resuspend the pellet in one milliliter of the nuclei suspension buffer and incubate on ice for 10 minutes.
After the incubation, centrifuge at 600 times g for 10 minutes. After the centrifugation, discard the supernatant, resuspend the pellet in one milliliter of the nuclei suspension buffer and centrifuge again. The final pellet will be the isolated nuclei.
Extract the proteins as described before adding 25 microliters of the 1X lysis buffer directly to the cell pellet. Then prepare two samples for SDS PAGE by taking 10 microliters each of the soluble protein extract and add 10 microliters of 2X Laemmli SDS sample buffer and one microliters of BME. Heat one sample at 37 degrees Celsius for five minutes and the second sample at 98 degrees Celsius for 15 minutes to reverse the formaldehyde cross-link.
Prepare one liter of 1X Tris-glycine running buffer by mixing 25 millimolar Tris, 192 millimolar glycine and 0.1%weight per volume SD.Then set up the SDS PAGE running apparatus. Open the 16%precast TGX SDS-PAGE package per the manufacturer's protocol and remove the cassette. Remove the comb that is lining the wells and the tape from the bottom of the cassette and place the gel into the running apparatus.
Fill the chamber with the 1X running buffer until the wells are submerged in liquid. Using a plastic pipette rinse out the wells with running buffer. Load the samples onto the gel along with 10 microliters of the pre-stained standard marker.
Finally, run the gel at 200 volts until the dye front is approximately one centimeter from the bottom of the gel. A western blot analysis of total cell extracts in the absence of BME reducing agent demonstrated a multimeric complex formation in different human cell populations. The complex disappeared with the addition of BME in all four cell lines examined, indicating the presence of a disulfide linkage.
The monomeric confirmation of dUTPase in human cells at the time of harvesting is a combination of at least three of the isoforms of dUTPase:the mitochondrial isoform, the nuclear isoform, and a truncated version noted at M24. The mitochondrial isoform used as a control did not form a disulfide linkage and migrated to the predicted molecular weight for the monomeric protein. Formaldehyde cross-linking of nuclear dUTPase demonstrated multimeric complex formation.
When the cross-link was reversed by incubating the sample at 95 degrees Celsius for 15 minutes, the complex was destabilized and could be visualized in its monomeric state. Adding iodoacetamide is an important step in this procedure to block the free cysteine residues and to ensure that the disulfide linkages are not a consequence of the extraction procedure. It is important to remember not to add any reducing agent, such as BME or DDT to your SDS-PAGE samples as these will reduce any disulfide linkages present in your sample.
