C-18Tips 蛋白质纯化方法

Purification and Concentration of Peptides Isolated From In-gel Digestion of Protein Bands by Zip-Tip As it would be in the case of Reversed-Phase HPLC, an important precaution to take during the procedure described below is never to allow the Zip-Tip column to dry out during any one of the steps. Channeling due to air-bubbles in the Zip-Tip will lead to poor retention of peptides, causing large losses of peptides, and also a great variability in yields of individual peptides. (The procedure described here may be used in parallel to process several digests side-by-side.) Items Required For Each Peptide Digest:

Zip-Tip C18 Cartridge column

Tube A. 100μ1 Acetonitrile (HPLC Grade) - Wetting Solution Tube B. 1000 μ1 water (HPLC Grade) - Cleaning Solution Tube C. 10-20 μ1 Sample Solution

Dissolve the extracted peptides in 10-20μ1 0.5% formic acid; make sure to wash the lower sides of the PCR tube containing the extracted peptides to achieve maximum dissolution of the peptides.

Tube D. 500 μ1 0.5% Formic acid - Desalting/Wash Solution

Tube E. 100 μ1 0.5% Formic Acid in 1:1 (v/v) water: acetonitrile - 1st Extraction Solution. Tube F. 100 μ1 Acetonitrile - 2nd Extraction Solution Tube G. A clean empty 200 μ1 PCR tube

1. Attach the Zip-Tip column to the 20μ1 micropipet (e.g. Gilson's Pipetman P20) snugly. Adjust the volume setting to 7μ1. Carefully, withdraw acetonitrile (Tube A) through the Zip-Tip and then, while the tip of the Zip-Tip is still under acetonitrile, pipet out the acetonitrile carefully, taking precaution to prevent introducing air bubbles into the Zip-Tip. Repeat this step at least 7-8 times, or until no bubbles arise during the pipetting out step. Finally, pipet out the acetonitrile slowly, and while the plunger is still down. immerse the tip of the Zip-Tip into the water (Tube B).

2. Slowly withdraw 7μ1 of water through the Zip-Tip, and then pipet it out carefully, taking care not to introduce any air into the Zip-Tip. Repeat this step carefully at least 10 times to ensure that all acetonitrile has been washed away.

3. Pipet out the water, and while the plunger is still down. move the tip of the Zip-Tip into the sample solution (Tube C). Carefully, fill the Zip-Tip with the Sample Solution, and, slowly push out into the tube (Tube C). Repeat at least 10 times to ensure that most of the peptides have been retained on the Zip-Tip.

4. In the same fashion as in steps 2, and 3, above, wash the Zip-Tip with 0.5% formic acid solution (Tube D) at least 10 times to perform the desalting and washing of the peptides.

5. After pipeting out the wash solution and. with the plunger is still down. transfer the Zip-Tip to the tube containing the extraction solution (Tube E), and slowly fill with the extraction solution. Wait for 20 sec to ensure a complete extraction.

6. Pipet out the extracting solution (Extract 1) into the empty tube G, and, while the plunger is still down. move to the tube F containing the acetonitrile. Slowly withdraw acetonitrile through the Zip-Tip.

7. After waiting for 10 sec., pipet out the solution (Extract 2) into the tube G containing the Extract 1.

8. Submit the combined extracts (Tube G) for MS Analysis, or store in a freezer until required for analysis. If you intend to us by mail, then we recommend that you dry the solution completely in a Speed-vac before mailing.

A Checklist For the Zip-Tip Protocol Reagent Digest 1 Digest 2 Digest 3 Digest 4 Digest 5 Wettins Step Repeat 7-8X, or until no air bubbles in the column Washine Step Perform washings 10X Sample Loadine Apply at -least 10X DesaltineA/Wash Perform at least 10 washings Extraction 1 ------------------- Extraction 2