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ZIPTIP Protocol
Notes
- Use C18 or uC18 to desalt peptides
- Use C4 ziptips to desalt proteins
- CHECK SAMPLE pH: MUST BE<3; Prepare solutions (below) fresh weekly
| Solution # |
Contents |
Application |
| 1 (wetting soln) |
50:50, ACN:H2O, 0.1% TFA |
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| 2 (wash soln) |
0.1% TFA in diH2O |
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| 3 (elution soln) |
60:40, ACN:H2O, 0.1% TFA |
for Peptides |
75:25, ACN:H2O, 0.1% TFA |
for Proteins |
Reconstitution solution
(for dried samples) |
5:95, ACN:H2O, 0.1% - 0.5% TFA |
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ACN = acetonitrile
TFA = trifluoroacetic acid |
Prepare three 0.6 ml Eppendorf tubes as follows:
| Tube labeled |
Contents of tube |
Application |
| waste |
empty |
|
| H2O |
100 ul of solution #2 |
|
| [sample info] |
1.2 ul of solution #3 (place on ice) |
uC18 ziptip |
| 2.5 ul of solution #3 (place on ice) |
C18 and C4 ziptips |
- Reconstitute samples (if necessary) with 13 ul of Reconstitution Soln and centrifuge; check pH
- Set a P10 pipettor to 10ul, place ZipTip on P10 pipettor
- HYDRATE TIP: Aspirate 10 ul of Soln #1 two times, discard each aliquot to waste
- HYDRATE TIP: Aspirate 10 ul of Soln #2 two times, discard each aliquot to waste
- LOAD sample: Aspirate 10ul of sample slowly and expel the sample back into the tube; Repeat this step 5-6 times.
- WASH: Aspirate 10 ul of water solution from H2O tube and expel into waste tube; repeat wash step 5-7 times
- ELUTE sample: Aspirate the Elution solution aliquot (this is the 1.2 or 2.5 ul aliquot of solution #3 in a 0.6 ml Eppendorf labeled with sample info) and expel back into the same tube. Repeat this 3 times (using the same aliquot of solution #3 each time).
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- Remember that the pipettor is set at 10 ul but you will only aspirate 1.2 or 2.5 ul at this step. Do not change the pipettor setting at this step otherwise you will introduce air into the packing material.
- Discard waste and H2O tubes
- Cap the sample tube and set on ice
- Add matrix (1 ul if elution volume was 1.2 ul; 2 ul if elution volume was 2.5 ul), mix, spot 1 ul of mixture on MALDI target
IMPORTANT:
- Pipet slowly to avoid introducing air into the packing material
- Do not aspirate air through the ziptip once the procedure has begun.
- If you must set the pipet down for some interruption, fill the tip with whatever solution you are currently working
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