The overall goal of this automated protocol for the serial dilution of peptide samples is to minimize peptide absorption to plasticware enabling accurate potency determination for drug discovery. This method can increase the quality and reproducibility of data and the removal of tip-based pipetting also eliminates sample carryover so that errors are not propagated throughout the entire serial dilution. The main advantage of this technique is that it increases data quality by minimizing peptide absorption to plasticware.
This is particularly important for driving a structure-activity relationship or lead identification campaign in drug discovery. After preparing the assay buffer and creating a dispensing program for a bulk dispenser according to the text protocol, immerse dispensing cassette tubing in assay buffer and prime with fluid. Place a 384 well low volume assay plate on a plate carrier.
Then press start. Dispense 25 microliters of 100 times peptide stocks into columns one through five of an acoustically qualified 384 well flat-bottomed polypropylene microplate to be designated as source plate A.Dispense 25 microliters of 100 times reference control into wells A23 and A24. Then dispense 10 microliters of assay buffer into columns 11 through 15 and 30 microliters of assay buffer into columns 21 through 22.
Next, into columns six through 10 and 16 through 20 of a second acoustically qualified 384 well polypropylene microplate designated source plate B, dispense 10 microliters of assay buffer. Centrifuge source plates A and B at 300 times g for one minute. Next, load the source and assay plates into the plate hotel section of the robotics.
Then running the robotics software according to the text protocol, use acoustic fluid dispensers integrated into the system to prepare three sequential one in 100 intermediate dilutions. Start with transferring from columns one to five on source plate A to columns six to 10 on source plate B.After preparing the first one in 100 dilution, allow automation to transfer source plate B to an integrated centrifuge and spin at 300 times g for one minute before returning the plate to the acoustic fluid dispenser. Then after transferring the solution from source plate B to source plate A for the second one in 100 dilution, automation will transfer source plate A to the centrifuge and spin before returning the plate to the fluid dispenser.
Finally, after automation transfers the solution from plate A to B for the third one in 100 dilution, plate B is again centrifuged. These peptide dilutions in source plate A and source plate B are then used to prepare full concentration response curves in the assay as described in the text protocol. This step is critical as a comparative tip-based methodology causes a dramatic loss in sample potency due to peptide absorption to plasticware.
Rapidly thaw cryopreserved CHO cells expressing the target mouse GLP1 receptor in a 37 degrees water bath and use 20 milliliters of assay buffer to resuspend them. Centrifuge the cell suspension at 200 times g and room temperature for five minutes. Discard the supernatant and use 10 milliliters of assay buffer to resuspend the cell pellet.
With Trypan Blue, dilute the cell stock one to one and use an automated cell counter to determine the viable cell density. Then use assay buffer to resuspend the cells at a concentration of 1.6 million cells per milliliter. Using a bulk reagent dispenser, add five microliters of cell suspension to each well of the assay plate containing serially diluted peptides and incubate at room temperature for 30 minutes.
To carry out the cyclic AMP detection assay, use the lysis buffer to prepare one in 20 dilutions of each homogenous time-resolved fluorescence or HTRF reagent. Use a bulk reagent dispenser to add five microliters of cryptate reagent to all the wells of the assay plates. Then add five microliters of D2 reagent to columns one through 22 of the plate.
Next, add five microliters of D2 reagent into wells E23 through P24. Immediately use a manual pipette to add five microliters of lysis buffer to wells A23 through D24 to be used as nonspecific binding control wells. Centrifuge the assay plates at 200 times g and room temperature for one minute to mix the wells.
Then cover the plates and incubate for one hour. After the incubation, use a plate reader to measure the fret signal at an excitation of 320 nanometers and an emission of 620 and 665 nanometers. Analyze the data according to the text protocol.
The first step in acoustic transfer for the cyclic AMP detection assay is to create four stock peptide intermediate dilutions across two source plates as shown here. For the second step, the acoustic dispenser further dilutes stock solutions from source plates A and B to create an 11-point concentration range for each test peptide in duplicate for 16 different peptides. Assay buffer backfill is performed to ensure a constant volume per well across the assay plate.
Shown here is a heat map for an example plate containing 16 peptides in duplicate run through the cyclic AMP accumulation assay. Low values shown in purple represent high cyclic AMP concentration. These concentration response curves illustrate the reproducibility of duplicate points.
Sample one represents a positive control for the assay. Samples two, three, and four were inactive and are represented by the flat lines on the graph. Samples five through 16 represent active test peptides.
The method presented here allows for the generation of full concentration response curves across a broad potency range. Once mastered, this technique can be completed in three hours, but this will depend on the number of samples requiring preparation. While attempting this procedure, it is important to remember that peptides are biological samples and should be handled as such.
In order to maintain their integrity, repeat freeze-thaw cycles should be avoided. This technique will pave the way for researchers in the field of peptide drug discovery and allow for the accurate potency predictions needed to fuel structure-activity relationships. After watching this video, you should have a good understanding of automated acoustic dispensing for the serial dilution of peptides and this will minimize peptide loss by absorption and increase the accuracy of your measurements.
Don't forget that working with biologically active peptides with picomolar potencies can be extremely hazardous and precautions such as personal protective equipment should always be used.
