The overall goal of this procedure is to discover protein interactions with immobilized bait molecules. This is accomplished by first acquiring and immobilizing the bait. The second step is to introduce the phage library to be screened to the bait under conditions promoting binding.
Next, the unbound phage are removed by stringent washing. While bound phage are used to infect the bacteria before being recovered. The final step is to amplify the bound phage for the next iteration of affinity selection.
Ultimately, when phage titer plateaus, single plaques are selected and sequenced to show which proteins have the highest affinity for the immobilized lagging under the experimental binding conditions. This process can help answer key questions in protein protein interactions. This procedure begins with purified recombinant protein being placed in the bottom of microtiter plates as described in the text protocol.
The next step is to inoculate 50 milliliters of Luria burani medium with 100 micrograms per milliliter of ampicillin in a 250 milliliter erlenmeyer flask with a single colony that has been previously grown as described in the text protocol incubate at 37 degrees Celsius, 200 RPM in a horizontal shaker and use a spectrophotometer to monitor cell density until an optical density at 600 nanometers of 0.5 to 0.6 is obtained. Then pour the cells into a 50 milliliter Falcon tube or similar and keep at four degrees Celsius until use cells will usually remain viable for approximately one week on day one. Prepare for affinity selection as described in the text protocol on the morning of day two.
Place nine autoclave empty, 250 milliliter erlenmeyer flasks in the clamps of an incubating shaker inoculate 500 milliliters of lal burani with 500 microliters from one of the overnight cultures of phage infected BLT 5 4 0 3 cells started the night before after placing the flask in the incubator, turn on the spectrophotometer and set it to read absorbance at 600 nanometers. Meanwhile, remove the microtiter plate from four degrees Celsius and take off the plastic film. Remove any unbound protein from the plate by washing 10 times with 200 microliters per well of one X tris buffered saline or TBS pH 7.5 and smacking the plate upside down on the paper towel between washes.
Block with 200 microliters per well of 5%blocking reagent in TBS for one hour. With the plate wrapped in plastic wrap, wash off the blocking solution 10 times with 200 microliters of one X-T-B-S-T smacking the plate upside down on four layers of paper. Towel between washes.
From here, use filter barrier tips to avoid phage cross contamination. Pipette 100 microliters of the phage library in with the proteins. Reseal the plate with plastic wrap and incubate at room temperature for one hour.
At the end of one hour, remove the plastic wrap from the plate and immediately wash by shaking out the phage into the biohazardous waste. Then use a multi pipetter to rapidly add 200 microliters of one X-T-B-S-T to each. Well set a timer for one minute.
After one minute, shake out the buffer into the biohazardous waste, smack the plate upside down on paper towel and place it back on the bench. Repeat washing steps 10 times after the 10th wash. Take 200 microliters of BLT 5 4 0 3 cells from the 50 milliliter falcon tube at four degrees Celsius and transfer to the bottom of each of the nine wells from which the last wash has been removed.
Wrap the plates in plastic wrap and put it 37 degrees Celsius for 20 minutes to get any phage still stuck to the bait to infect the bacteria at the end of 20 minutes. Unwrap the plates. Next, remove 10 microliters of bacteria from the BSA at 25 degrees Celsius replication one well and transfer to the 990 microliters of medium marked one.
Repeat this process twice more for that well resulting in three triplicates of one to 100 dilu of the phage from that well this is BSA replication one sampled three times. These dilution will be used to estimate the average titer plus or minus the standard error of the mean for that replication of BSA do the same for replication two and three of BSA for the three replicated wells of the poisoned bait protein and for the bait protein set these 27 EOR tubes aside. If the bacteria in the flask are at an optical density of 0.6 to 1.0 decant 50 milliliters of the cells from the fern flask into each of the 9 250 milliliter erlenmeyer flasks.
Take the remaining 170 microliters of phage infected BLT 5 4 0 3 bacteria in the BSA replication one well and add it to the 50 milliliters of cells marked BSA rep.One. Do this for all nine wells before shaking the flasks in the 37 degrees Celsius incubator. Meanwhile, perform dilutions from the initial 27 dilutions by taking 100 microliters of the LB with bacteria from Einor tube one and adding it to the 900 microliters of LB in einor tube.
Four for the 10 to the minus third dilution, discard the filter barrier tip for a new one before getting 100 microliters of the LB with bacteria from einor tube four and adding it to the 900 microliters of LB in eph tube. Seven for the 10 to the minus fourth dilution, continue the dilution for all triplicates of all replications of all proteins and treatments. There should be 135 tubes in total once the cells have lyed.
Bring the culture to 0.5 molar sodium chloride by adding five milliliters from a five molar sodium chloride stock and transferring the sample to a labeled centrifuge bottle centrifuge at 8, 000 times G for 10 minutes at four degrees Celsius. Then decant the supernatant containing the virus into a clean, sterile 50 milliliter falcon tube. Add a few drops of chloroform to the decanted supernatant in the Falcon tube.
The supernatant can be stored at four degrees Celsius for the next round of affinity selection on day three, pipette 250 microliters of VLT 5 4 0 3 cells from four degrees Celsius into each of the previously prepared numbered bo silicate test tubes. Then pipette 100 microliters from the dilution in einor tube one into the 250 microliters of cells in bur silicate test tube.One. Briefly heat the first five inches of the pipette over the flame and plunge it into the molten top aros.
Pull up three milliliters and deliver quickly into the test tube on top of the cells and phage. Mix by flicking with a finger while holding the tube with the other hand, and then pour the contents on the plate. Tilt the plate and use the test tube to chase bubbles and dry spots until the whole plate is covered by the top agros.
Set it aside upright on the bench to cool. Discard the boros silicate tube. Eject the filter barrier tip and get a fresh one and continue until all the dilution have been plated.
Retrieve prepared plates from the incubator as they're depleted, but no more than six at a time or they cool and the top agros solidifies too quickly, examine the plaque formed on the plates and discard those with confluent lysis. Determine which of the remaining serial dilutions have produced sufficiently few plaques to enable an accurate Cali Record the number of plaque from these plates and proceed to calculate titer as described in the text protocol at the end of affinity selection. Round four, select 18 individual well-defined plaque colonies using a sterile yellow pipette tip.
Wet the inside of the tip with tris hydrochloride pH 8.5 in an einor tube. Then core these plaques from the tittering plates by pushing the tip through a well isolated plaque directly to the plastic Petri dish below and aspirating the core, expel the core into 100 microliters of tris hydrochloride pH 8.5 in the appropriate tube before vortexing. Briefly, heat 20 microliters from this volume at 65 degrees Celsius for 10 minutes and proceed with PCR and sequencing as described in the text protocol shown here are typical titer results by sampling several times the final estimated tallies are not as susceptible to pipetting errors and a more accurate estimate of the actual titer and the variation around this.
Estimate well to well are obtained increasing phage titer preferentially for the non poisoned bait as the number of affinity selection rounds increase. Also provides confidence that the technique is working. The retention of an increasing percentage of phage containing insert in non poisoned bait wells as the number of affinity selections increases is also auspicious after advanced rounds of affinity selection.
An increase in the number of independently recovered phage that have insert relative to the first round and the settling of these amplicons on a few identically sized bands is a good indication that particular clones are being selected in the affinity selection Following this procedure. Other methods like ease two hybrid can be performed in order to validate the interactions discovered by phage display.
