Tuesday, February 15, 2011

Zip Tip Protocol~ C18 tips

The zip tip protocol is used to clean up the biological samples after protein digestion.  Its main purpose is to get rid of the chemicals used in protein digestion, such as DTT, IAA, salts, SDS, etc.

Three solutions need to be prepared in order to proceed with the procedure: solution A, solution B, and solution C.

Solution A: Rinse Solution (0.1% formic acid 100%)
Final volume:1000 ul
(100%)V=(0.1%)(1000 ul)
V=1 ul FA 100%
*Obtain 1 ul FA 100%, 999 ul of water*

Solution B: Wet Solution (0.1% FA 100%, 50% acetonitrite)
Final Volume: 1000 ul
*Obtain 1 ul FA 100%, 500 ul ACN, 499 ul water*

Solution C: Elution Solution (0.1% FA 100%, 60% ACN)
Final volume: 1000 ul
*Obtain 1 ul FA 100%, 600 ul ACN, 399 ul water*

(Added 70 ul of water to the dried biological samples)
1. Set pipette to 10 ul.
2. Equilibrate tip with wet solution B.  Aspirate wet solution into the tip.  Dispense to waste.  Repeat twice.
3. Aspirate rinse solution A.  Dispense to waste.  Repeat once.
4. Bind peptides.  Aspirate and dispense the sample 10 to 20 times (depends on sample complexity).
5. Aspirate rinse solution A and dispense to waste.  Repeat 7 to 10 times.
6. Elute peptides.  Set pipette to 5 ul.  Aspirate 5 ul of elution solution C into the tip and place in a clean vial.  Repeat once or twice.
Dilute to desired final concentration with spraying solution: 0.1% FA, 50% ACN (never actually made this because for my lab it is unnecessary, my mentor and I just dried up the samples again after these steps)