The overall goal of this experimental protocol is to enable inexperienced PTM investigators to effectively determine if their target protein is endogenously modified by one or more PTMs using a single comprehensive system. This method will enhance investigation into post translational modification field by enabling detection of key PTMs and potential crosstalk for any target protein using a single, simplified system. The main advantage of this technique is that non-PTM experts can detect whether a protein is endogenously modified by one or more PTMs in their model system.
We first had the idea for this method while studying regulatory mechanisms for several cell signaling pathways, and discovered our user-friendly kits or systems were not really available for studying non-phospho PTMs. Begin this procedure with preparation of A431 cells, as described in the text protocol. Also, prepare blaster lysis in dilution buffers with inhibitors.
Pour off the culture media, and place the cells on ice. Then wash the cells twice with 10 milliliters of 1x phosphate buffered saline. Remove as much PBS as possible prior to adding blaster lysis buffer to maximize cell lysis.
Add 600 microliters of blaster lysis buffer to each 150 millimeter plate. The amount of buffer is based on expected protein yield. Next, lyse the cells using a cell scraper.
The lysate will become viscous due to nuclear lysis. Use a snipped one millimeter pipette tip to transfer the crude lysate into a blaster filter that has been placed in a 15 milliliter collection tube. Use the supplied filter plunger to completely compress the blaster filter, and collect the lysate flow-through, including any bubbles, in a 15 milliliter tube.
The simple, efficient blaster filter removes genomic DNA, rather than shearing it as seen in other commonly used methods. Dilute the clarified lysate with blaster dilution buffer to a final volume of three milliliters for each 150 millimeter plate. The final volume is based on expected protein yield.
This step is important, as the final buffer composition will affect the amino precipitation reaction's stringency. Next, prepare the protein quantitation assay, as described in the text protocol. Measure absorbance of the lysate sample at 600 nanometers, and calculate the protein concentration.
Invert the stock reagent tube several times to make sure that the beads are completely resuspended in the tube. For each IP assay, aliquot the bead suspension into separate 1.5 milliliter micro centrifuge tubes on ice. Here, add 20 microliters of ubiquitin affinity beads, 30 microliters of phosphotyrosine affinity beads, 40 microliters of SUMOylation 2/3 affinity beads, or 50 microliter of acetylation affinity beads to the individual micro centrifuge tubes.
Invert the stock reagent tubes containing ubiquitination IP control beads and IGG control beads several times to make sure that the beads are completely resuspended in the tube. Next, aliquot the control beads per control reaction to determine nonspecific binding. Here, add 20 microliters of ubiquitin control beads, 30 micro liters of phosphotyrosine control beads, 40 microliters of SUMOylation 2/3 control beads, or 50 microliters of acetylation control beads to individual 1.5 milliliter micro centrifuge tubes on ice.
Save 20 microliters of lysate to run as a western blot input lysate control. Add five microliters of 5x sample buffer to the reserved lysate and boil at 95 degrees Celsius for five minutes. Next, add lysate to each IP tube and control IP tube.
Here, one milligram of lysate was used per IP and control reaction, resulting in a total of eight IP reactions. incubate the tubes on an end over and rotating platform at four degrees Celsius for two hours. After incubation, collect the beads by centrifugation at three to 5, 000 times G for one minute at four degrees Celsius.
Aspirate off as much supernatant as possible without disturbing the beads, then wash the beads in one milliliter of blaster wash buffer for five minutes on a four degree Celsius rotating platform and collect the beads by centrifugation as before. Following centrifugation, aspirate off as much supernatant as possible without disturbing the beads and repeat the wash step two more times. After the final wash and centrifugation, aspirate the majority at the buffer supernatant, then remove the residual supernatant using a fine bore protein loading tip.
Minimal disruption of the bead pellet is acceptable. Ensure that all wash buffer has been removed while minimizing disruption to the beat pellet in order to maximize recovery of your PTM proteins. Next, add 30 microliters of bead elution buffer and resuspend the beads by gently tapping or flicking the side of the tube.
Do not use a pipette at this stage. Incubate the resuspended beads at room temperature for five minutes. Snip the end off of the transfer pipette tip and gently transfer each bead suspension to a 1.5 milliliter micro centrifuge spin column that has been placed in a 1.5 milliliter micro centrifuge tube.
Centrifuge the sample at between nine and 10, 000 times G from one minute at room temperature to collect the IP sample, then add two microliters of 2-mercaptoethanol to each sample and mix well. Place the samples in a 95 degrees Celsius water bath for five minutes. Following incubation, collect the sample by centrifugation at 10, 000 times G for one minute at room temperature.
Finally, proceed to running SDS PAGE, transfer, and western blot analysis as described in the text protocol. Untreated and EGF treated A431 cell lysate was added to ubiquitin, phosphotyrosine, SUMO 2/3, and acetyl lysine affinity beads. A PD-L1 antibody was utilized for western immunoblot analysis to determine if the PD-L1 protein was modified by these four PTMs in response to EGF stimulation.
Representative results shown here indicate that this method effectively identifies endogenous PTM modifications of PD-L1. Once mastered, this technique can be done in four to 4 1/2 hours if performed properly. After watching this video, you should have a good understanding on how to detect enogenous PTM or crosstalk for your target protein or signaling pathway by using this comprehensive PTM detection system.
