Instructions for Setting up the Plate
The print plate provides the foundation for a protein microarray experiment. It represents the direction all experiments will follow and the layout of the proteins on each slide. The act of setting up a plate is also a big chunk of work so it's worth taking some extra time to do it right.
Plan out the Plate
Once the peptides/proteins have been obtained and reconstituted at the optimal concentration (ours is 0.2mg/mL), it's time to plan out the plate. The exact location of each protein is not crucial as long as you know where it is.
The 384-plate might seem overwhelming because there are so many wells. Just how can one stay focused to add the approximately 200 (remember we add duplicates, at least, of each protein to a plate) unique proteins?
Below are some suggestions that will make the plate making more successful
- Add positive and negative controls to the plate. We use fluorescent markers (antibodies), vaccines and epitopes known to be reactive for our positive controls. We use PBS or different antibodies from different species as negative controls.
- Distribute controls uniformly throughout the slide. It's a good idea for A) checking the uniformity of the surface and B) ensuring that one bad area of the slide doesn't invalidate the entire experiment.
- Add at minimum of four replicates of each unique protein. There is a trade-off between maximizing the number of unique proteins on the slide and being able to obtain meaningful results from the values. Each spot will inevitably have variation in the number of molecules that bind to it. Four replicates ensures that one can do statistics to determine an average or median value that reflects an accurate value for molecules that are interacting with the unique protein.
- Make up one plate and print it twice. One plate reduces the work and cuts in half the amount of peptides one has to keep track of.
- Make up multiple replicates of a plate in any given print making session. This minimizes the freeze-thaw cycles for the proteins and maximizes the setup effort. It takes a fair amount of planning and organization to put together a print plate. The time to make a few more replicates in one session is small in comparison to the time to make a plate before every print. Besides, it's just much better to spend a few hours of making plates and not have to worry for another 6-12 months.
- Mark off the 384-well-plate into X by X grids (where X by X represents the number of pins used to print). We print with 16 pins in a 4 x 4 configuration so I mark off the 384-well-plate into 4 x 4 grid.
- 7. Have someone help you. It's great to have a helping hand for maintaining sanity, double checking your work, handing you the proper vial, or having a nice conversation. One can always bribe a person with lunch if one's lab mates are not so altruistic.
8. Organize the aliquots into the layout of the plate.
9. Tape the edges of the plate down to the table to prevent the plate from moving when using the pipette.
10. Add 7mL - 8mL of the protein in its optimal concentration per well. We can usually get two to three prints (each print means that the plate is duplicated) with this volume.
11. Label each plate. The date the plate was made and the number (1 of X) is fine.
12. Seal the plates and store them in the -80oC freezer. We seal the plates with aluminum tape and place them into heat seal bags before placing them into the -80oC freezer.