ProteinSimple eBook - 17

OPERATION

LIQUID

VOLUME
(ML)

Flush

Filtered water or filtered buffer

0.90

Flush

Filtered water or filtered buffer

0.90

Flush

Filtered water or filtered buffer

0.90

Dry system
Flush

Filtered water or filtered buffer

0.90

Optimize
Illumination

Filtered water or filtered buffer

0.22

Baseline

Filtered water or filtered buffer

0.70

Flush

Filtered water or filtered buffer

Method Transfer
Translating a manual method to an automated method requires modiﬁcation of the protocol to ensure
comparable results due to differences in sampling handling between the two methods.

0.90

Dry system
Stir 4 cycles, speed
5

Sample (well 1)

0.90

Flush

Sample (well 1)

0.50

Stir 4 cycles, speed
5

Sample (well 1)

0.90

Optimize
Illumination

Sample (well 1)

0.22

Stir 4 cycles, speed
5

Sample (well 1)

0.70

Analysis Run

Sample (well 1)

0.70

Flush

Filtered water or filtered buffer
Filtered water or filtered buffer

0.90

Flush

Filtered water or filtered buffer

0.90

Method transfer considerations include:
* Changes in sample introduction-manual pipetting or stirrer with syringe barrel mix differently than an
automated pipettor. The user can control the duration and speed of mixing with the automated pipettor.
* Suitability of sample type-manual or automated sample requirements
are similar; highly viscous or extremely concentrated samples may not be suited for automation.
* Sample volumes-sample and purge volumes may change due to new
labware and ﬂuid path.
* Ambient temperature-samples should be stable at room temperature
during an automated run.

0.90

Flush

Table 1. Description of automated Protocol D
used for 1% BSA solution. Original version of
Protocol D uses 0.90 mL for Flush step. Steps from
Flush after Baseline through Analysis Run are
repeated for each sample in the run. Stir of four
cycles was used prior to each sample analysis to
ensure particles remained in suspension.
17

Simplifying Protein Analysis * Comparability Study of Manual and Automated Particle Characterization with MFI

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Sample volumes should be chosen based on particle concentration in the sample, as described in the next
section. Sample volumes should also be adjusted further to accommodate the sample delivery format of the
instrument conﬁguration. Thus, moving from a manual to automated protocol may require changes to sample
volume and ﬂush volume initially to achieve accurate results. This is due to the ﬁxed sample delivery format
which includes 1-2 mL deep well plates, the 1 mL pipette tips and the interface to the inlet port (Table 2).
Optimization of the automated protocol for the 1% BSA solution was performed as follows. We used a
larger volume initially in Protocol D and then modiﬁed pipetting parameters as described in the modiﬁed
Protocol D to reduce the volume requirements (Table 2). Additional stirring steps counteracted the effects of

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ProteinSimple eBook

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