The procedure for screening peptides.

the first
, a target molecular solution of 100 mg/ml is prepared at 0.1M NaHCO3 (pH 8.6). If the target molecule needs to be stabilized, a similar ion strength buffer containing metal ions can be used.
add 1.5 ml of target molecular solution to each hole and repeat the vortex until the surface is completely moist (this step requires more attention to avoid the solution forming the beads).
in a wet box, a gentle oscillation of 4 degrees C incubates overnight. Save in a wet box at 4 degrees C.the next
, the ER2537 (bacteria
cultured
, which was used to lay the board during the titration determination) was inoculated into a 10 ml LB
medium
. If the
phage
is amplified on the same day, the ER2537 overnight culture can also be inoculated in a 250 ml conical bottle containing 20 ml LB medium (1:100 ratio). Shakes violently at 37 degrees C.
the flat dish back on a clean paper towel, remove the bag of liquid, fill it with a closed liquid, and incubate at least 1h at 4 degrees C.
removal package is liquid. Wash 6 times quickly with TBST (TBS plus 0.1% Tween-20). Repeated rotation ensures that both the bottom of the hole and the side of the hole are washed. Remove the washing fluid according to step 5 (it can also be washed with an automatic washer). Wash quickly and avoid drying
flat
. (Note that the paper towels are replaced each time to avoid cross contamination).
1 ml TBST to dilute 4' 1010 phages (10 ml original library), add to the flat dish after the package, gently shake at room temperature 10 to 60min.
remove unorthroted phages in step 5.
to step 6 method with TBST washing board 10 times, each time to replace clean paper towels, to avoid cross-contamination.
the phages that bind to 1 ml of wash-out buffer. The semoulation can be TBS containing ligations (0.1-1mM) or TBS containing target proteins (-100mg/ml) to compete with target proteins cured on flat dishes to bind to phages. Gently shake 10 to 60min at room temperature. Transfer the semen into a trace
centrifugal tube
tube.
10a. or use a universal buffer, 0.2M glycine-hydrochloric acid (pH 2.2), 1mg/ml BSA. Gently shake no more than 10min and transfer the wast into a micro centrifuge tube to balance 150ml 1M Tris-HCl (pH 9.1).
a small amount of sequestration (-1 ml) to determine the titration, if necessary, the first or second round of titration of the phage can be sequenced (see follow-up operation). (Unused secrubbing can be stored overnight at 4 degrees C and amplified the next day.) In this case, ER2537 is cultured overnight with LB, and the next day, the culture is diluted to 20 ml with 1:100 with LB culture, an unexpanded semen is added, and in a conical bottle of 250 ml, 37 degrees C shakes and incubates 4.5h vigorously, entering step 13).
the remaining carcides are amplified: the carcations are added to 20 ml ER2537 cultures (early in the number of years), and 37 degrees C shakes violently to incubate 4.5h.
the culture into a trace centrifuge tube, 4 degrees C, 10,000 centrifuges 10min, the upper qing into a new centrifuge tube, centrifugal again.
80% of the upper clean into the new centrifuge tube, adding 1/6 volume of PEG/NaCl. Precipitate phages at least 60min or overnight at 4C.the third day
4 degrees C, 10,000 centrifugal PEG sediment 15min, dumped on the clear, centrifuged again, sucked away the excess on the clear.
1 ml of TBS suspended sediment and transferred to a trace centrifuge tube, 4 degrees C centrifugation 5min to precipitate the remaining cells.
will be transferred to a new centrifuge tube, with 1/6 volume of PEG/NaCl re-precipitation, ice incubation 15 to 60min. Centrifuge 10min at 4 degrees C, discard on the Clear, once again briefly centrifuge, with a trace of the gun head to suck away the remaining upper Qing.
200ml TBS, 0.02% NaN3 suspended sediment, centrifugal 1min, will be transferred to the new tube, that is, amplified semen.
to determine the concentration of the secum after amplification and stored at 4 degrees C.
the bag is flat dish for the second round of screening, the same step 1 to 3. the fourth and fifth days
count blue phage spots, determine the titration of the phage, and calculate the volume of the phage corresponding to 1-2'1011 pfu. If the titration is too low, the phage volume corresponding to 109 pfu can be used for screening.
second round of screening: repeat steps 4 to 18, with the first round of amplification of phage containing 1-2'1011 pfu phage screening, the concentration of Tween 20 in the washing liquid increased to 0.5%.
the titration of the second round of screening amplification semen.
is flat dish for the third round of screening, steps 1 to 3. the third round of screening on the sixth day
: screening with 1-2'1011 pfu phages in the second round of amplification semen, the concentration of Tween20 was still 0.5% when washing.
to determine the titration of the un amplified third round of semen. If you do not perform the fourth round of screening, you do not have to amplification of the third round of screening seides. Blue phage spots at titration can be used for sequencing: plate incubation times must not be longer than 18h, as too long may cause a decrease in phage infection rates. Keep the remaining escapes at 4 degrees C.
selected monoclonal ER2537 overnight culture.
.