| 1. |
What is the difference between the OMIX C18
and C18MB tips? |
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Both the C18 and the C18MB OMIX pipette tips utilize
a 10 µL tip housing and contain a C18 stationary phase.
The only difference between the two configurations is the quantity
of material packed into the tip. The OMIX C18MB (MiniBed) version
contains ~1/3rd the sorbent material to achieve lower elution
volumes (0.5-2 µL) required for maximum enrichment and
improved sensitivity. OMIX C18 tips are recommended for applications
that require higher capacity and elution volumes in the 2-10
µL range. Note: all solvent volumes utilized in the OMIX
procedure, other than the final elution step, are the same for
the C18 and C18MB configurations. |
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| 2. |
What types of peptides and proteins can be
purified with the OMIX product? |
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OMIX tips contain a reverse-phase C18 functional
group on a monolithic silica surface. Any peptide or low molecular
weight protein with some hydrophobic nature will have a strong
affinity for the OMIX sorbent. The monolithic properties of
the silica layer in OMIX provide higher binding capacities than
other tip materials since maximum exposure to the functionalized
surface is achieved with the uniform flow through the tip. |
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| 3. |
What types of interferences will be removed
with the OMIX product? |
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OMIX pipette tips will efficiently remove salts,
detergents and hydrophilic contaminants that do not bind to
the reversed-phase media. The majority of these contaminants
will be eliminated during the sample application step. Any weakly
bound interferences remaining on the OMIX sorbent surface during
sample application can be easily removed in the rinse step with
a 0.1% trifluoroacetic acid (TFA) buffer. |
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| 4. |
What if there is no flow through the tip? |
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Varian tests each lot of OMIX tips to ensure that
your product meets our stringent flow specifications. Thousands
of OMIX tips have been tested with a <0.01% plugging rate.
Once the tip has been conditioned with the acetonitrile/water
or methanol solution, it is critical to keep the membrane wetted
to ensure proper binding of your analytes to the OMIX sorbent.
If air is drawn into the tip before the sample application,
you may experience slow to no flow that will result in inconsistent
and low recoveries of your peptides. If the OMIX sorbent is
exposed to air before sample application, aspirate and dispense
with two additional aliquots of the conditioning reagent to
re-wet the tip.
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| 5. |
How do I ensure optimum aspiration with less
than the full 10 µL volume? |
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Always leave the pipettor set to 10 µL.
A full 10 µL volume is recommended for all solutions throughout
the OMIX procedure EXCEPT for the final elution volume when
lower quantities are required for peptide enrichment. Optimum
recoveries during the final step are obtained when the elution
reagent is pipetted into a separate vial or well plate. Then,
with the pipettor set to 10 µL, carefully pipette the
entire volume of elution solvent into the OMIX tip. It is not
recommended to aspirate and dispense the sample more than once
during the elution step. |
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| 6. |
Why is trifluoroacetic acid (TFA) the preferred
acid for the sample pre-treatment and elution solvent? |
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TFA is commonly used as an ion-pairing agent in
peptide and protein analysis for sample cleanup as well as high
performance liquid chromatography (HPLC) applications. In an
acidic environment, any basic residues on the peptide will form
a positive charge. The negative counter ion in the acid will
then pair with the positively charged base. Using a counter
ion with a small organic chain, like TFA, will increase the
hydrophobicity of the peptide and increase the affinity of the
peptide for the hydrophobic stationary phase. |
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| 7. |
What other acids can be used with the OMIX
tips instead of TFA? |
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For analysis by LC/MS or LC/MS/MS, the TFA in
the elution solvent only should be replaced with either
0.1% Formic Acid or 0.1% Acetic Acid to avoid ion suppression
caused by TFA. Both the formic and the acetic acid can be used
with a 50-75% acetonitrile solution for optimum recoveries.
Note: to ensure maximum binding of peptides to the OMIX sorbent,
the TFA solution should always be used in the pretreatment step.
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| 8. |
How can the recovery of hydrophobic peptides
be improved? |
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Insufficient disruption of the binding between
the OMIX sorbent and the peptide during the elution step is
the primary cause for lower recovery of hydrophobic peptides.
Increasing the acetonitrile concentration in the elution solvent
to 75-90% will improve the release of these hydrophobic peptides.
If elution is being performed with 0.5-2 µL of elution
solvent, do not re-aspirate the elution solvent to prevent the
hydrophobic peptides from re-binding onto the membrane. |
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| 9. |
How can the recovery of hydrophilic peptides
be improved? |
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To ensure best results of samples that contain
hydrophilic peptides or a wide range of peptides, increase the
TFA concentration to at least 1.0% in the sample pretreatment
step. |
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| 10. |
Can the OMIX Tips be reused? |
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No, we do not recommend reusing the OMIX tips
to eliminate carryover and sample contamination problems and
ensure maximum recovery of your target peptides. |
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| 11. |
What pipettors can be used with OMIX? |
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OMIX tips are based on a standard 10 µL
tip format and is compatible with most P10 single channel and
multi-channel pipettors. The brands of pipettors with confirmed
compatibility on the OMIX tips includes Eppendorf Research,
Gilson P10, Rainin, Oxford, Finnipipette multichannel, VWR single
and multichannel pipettors. |
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| 12. |
Can OMIX pipette tips be used with robotic
systems? |
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Yes. Both the OMIX pipette tip and tray that houses
96 OMIX tips have been tested for automation compatibility on
several robotic systems. OMIX presently has the only 10 µl
tip rack that has been designed to be consistent with the Society
of Biomolecular Screening (SBS) specifications. The SBS footprint
is standard for all liquid handling systems. For an up-to-date
list of OMIX recommended robotics vendors, email omix@varianinc.com
or contact your local Varian representative. |
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