Difference: MicroDialysisPlatesExperiment (14 vs. 15)

Tests of the micro-dialysis plates performances

Reference

Experiments; test of the plate

June, 23rd 2011 meeting - Tests of the performance of the plate

• Summary of Ralph's presentation:
  • calibration curves done with fluorescein
  • tested with cassettes: after dialysis OD~0.12 as expected (after 46h)
  • tests in the micro-dialysis plate:
    • OD at 280nm observed in the buffer chamber, even if only water is placed >> nylon 6 contamination from the bottom of the chamber. Tried to remove the peak of the contaminant on the curves.
    • dialysis seem ok
    • no leakage observed
    • no cross-contamination observed
    • evaporation may be problematic (and/or pippetting >> results not exactly similar in every tested wells)
    • dilution may occur if levels in the two chambers are different
• Comments:
  • If curves are saturated, dilution must be done
  • Does the nylon 6 contaminant enter the sample chamber?
  • BSA can be followed by SDS-page
  • Preparation of the dialysis membranes? Ask Jiang if a preparation is done. Ask Tamir for its protocol
  • Alu foil only covers the plate; it may need to be sealed.
    • Changki: Depending on the seal chosen, you may not be able to use the robot any more
    • Ralph: Would a seal loosen the membrane? To be determine.
    • Nathan suggests o-ring like sealing on a cover which could be easily removed.
  • if level in the sample chamber is lower than in the buffer chamber, level will come to an equilibrium and a dilution will occur

May 2011

April 2011

Sept 2010 (Martin's archive)

July 2010 (Martin's archive)

The performance of the minidialysis plate was evaluated by quantifying the rate of detergent removal through thin layer chromatography (TLC) and by producing crystals of the membrane protein P2A3.

For the detergent removal tests, three detergents with very different CMC’s were evaluated: 100mg/ml OG (CMC: 5.3 mg/ml), 30mg/ml DM (CMC: 0.87mg/ml), and 20mg/ml DDM (CMC: 0.087mg/ml).

OG was rapidly eliminated from the dialysis wells, and after six buffer exchanges all OG had been removed. Interestingly, the rate of dialysis seems to be a function of time, and independent of buffer exchange frequency (compare figures below). Thus, the gradient difference over the dialysis membrane seems not to be the rate limiting factor for OG removal from the sample well, since frequent buffer exchanges do not increase that rate. In fact, the rate of removal is essentially linear with time for the first 40h of dialysis or until approximately 80% of the detergent has been removed, whereafter the rate slows down considerably. These results indicate that the rate of dialysis may be governed by some intrinsic properties of OG, such as its interaction with the dialysis membrane that may reduce its diffusion speed, or disassembly of the OG micelles.

OG removal with the micro-dialysis plate

DM was removed from the dialysis wells at an intermediate rate. After around 10 buffer exchanges or 150h, almost all of the detergent was removed. Note that also for DM the rate seems to be independent of the buffer exchange frequency, which is especially evident for buffer exchange no 7 where the buffer was left in the plate for close to 40h before it was changed, and therefore allowed a lot of DM to be removed at once.

Also in this case the there seems to be another rate-limiting step to the speed of removal than the frequency of buffer exchanges, since also here the initial part of the removal curve is essentially linear when plotted against time but not when plotted against buffer exchange number.

As expected, DDM was removed considerably slower than the other detergents and required roughly 25 buffer exchanges and a total dialysis time of two weeks.

DDM removal with the micro-dialysis plate

These results are similar to the results obtained with the crystallization block (Vink et al., 2007) and confirm that even detergents with very low CMCs can be consistently removed through dialysis.

Next, the protein P2A3 (purified in DDM) was dialyzed in the minidialysis plates using conditions previously established with the larger dialysis block and with dialysis buttons. After two weeks of dialysis, with buffer exchanges twice per day, long tubular crystals were observed with a similar abundance to those obtained with the other dialysis devices. These results clearly demonstrate the functionality of the microfluidic dialysis plate and work is currently underway to incorporate the plate into our crystallization pipeline.

-- NicolasCoudray - 20 Jan 2011

* ralph_august25.ppt: cyclodextrin+microdialysis (YiiP^?)