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ZipTipC18 tips- a standard bed of 0.6 uL for sample elution in 1 to 4 uL. Appropriate for peptides and low molecular weight proteins.
ZipTipC4 tips- a micro bed of 0.2 uL for sample elution in <1 uL. Appropriate for low to intermediate molecular weight proteins, proteins over 100,000 Da.
The procedure also requires a compatible 10 uL pipettor. For simultaneous processing of multiple samples, Millipore recommends the Biohit Proline™ Multi-channel Pipettor.
|Solutions (make fresh daily):||ZipTipC18 tips||ZipTipC4 tips|
|Wetting solution||100% Acetonitrile (ACN)||100% Acetonitrile (ACN)|
|Sample Prep||0.5% TFA in ddH2O||0.5% TFA in ddH2O(optional) Guanidine-HCl|
1-6 M maybe added
|Equilibration solution||0.1% TFA in ddH2O||0.1% TFA in ddH2O|
|Wash solution||0.1% TFA in ddH2O||0.1% TFA in ddH2O|
|Elution solution||0.1% TFA/ 50% ACN|
With or without Matrix
|0.1% TFA/ 50%ACN|
With or without Matrix
Maximum binding occurs in the presence of TFA (0.1%) or other ion-pairing agents. To maximize analyte binding, use the appropriate sample preparation solution. Ensure that the final solution has a pH < 4.
Optimal binding may require a chaotropic agent (guanindine-HCl at a final concentration of 1-6 M). If the sample does not already contain chaotropic salts, add them a few minutes prior to binding. These salts will be removed during the wash step following binding.
Equilibrate the ZipTip pipette tip for sample binding
Bind and wash the peptides or proteins
Follow these steps after equilibrating the ZipTip pipette tip:
NOTE: 5% methanol in 0.1% TFA/ water wash can improve desalting efficiency. Additional washing may be required for electrospray MS.
Elute the peptides or proteins
For ZipTipC18 (standard bed format) and ZipTipC4, pipette tips, dispense 1-4 uL of elution solution into a clean vial using a standard pipette tip. In the case of ZipTipu-C18 (micro bed format) pipette tips, dispense 0.5-2 uL of elution solution into a clean vial.
Caution: Acetonitrile and methanol are volatile and evaporation can occur rapidly. If this occurs, add more eluant to recover sample.
Carefully, aspirate and dispense eluant through ZipTip pipette tip at least three times without introducing air into the sample. Sample recovery can be improved (at the expense of concentration) by increasing the volume to 5 uL.
For direct spotting onto a MALDI-TOF MS target
Elute with or without matrix elution solution.
Prepare the following solutions:
These are Final concentrations; I suggest that you prepare a 50 mM stock of ABC to start with.
25 mM NH4HCO3 (100 mg/50 mL) (ABC)
25 mM NH4HCO3 in 50% ACN
10mM dithiothreitol (DTT) in 25 mM NH4HCO3 (1.5mg/mL)
55mM iodoacetamide (IA) in 25mM NH4HCO3 (10mg/mL)
15 ng/mL trypsin in 25 mM NH4HCO3 (freshly diluted)
For low-level proteins (<1 pmol), especially those separated by 1-D SDS-PAGE, reduction and alkylation is recommended. These procedures are performed after step 5.
Extraction of Peptides
Sample is now ready for mass spectrometric analysis.