HTD96b is our standard model. The HTD96c version was designed to reduce the time required to reach equilibrium by decreasing the diffusion path length and increasing the dialysis membrane surface area to sample volume ratio. The reduced well volume design minimizes assay sample volumes (25-75ul) to reduce reagent costs. The smaller well requires greater precision when pipetting. Both the HTD96b and HTD96c Teflon blocks use the same base clamp so if you purchase one model and decide to switch, you only need to purchase the alternative Teflon block rather than another complete unit.

The dialysis block can be incubated at any desired temperature between 20ºC and 45ºC. Equilibrium is reached more rapidly if the dialysis block is shaken during the incubation period. Shaking at 80-100rpm is sufficient using any general incubator containing an orbital or reciprocating platform shaker e.g. Fisher Scientific (Catalog # 14-278-104) and VWR international (Catalog # 47742-750 or #33998-360).

Use a colored marker pen and color the blocks over and around the letters. The color generally does not penetrate the letters but does color the surface. After allowing time to dry, thoroughly wipe the surface thereby removing some of the color. This usually results in a faint coloration that contrasts with the white of the letters making them more legible. This color will remain through several washes and should facilitate reading the letters. Another advantage is that if you use different colors for each block you have also quickly identified the correct bars for each set if they get mixed during washing.

Dialysis membranes consist of a matrix of cross-linked polymers. The pore rating, Molecular Weight Cut Off (MWCO), is an indirect measure of the retention performance using a series of standard molecules with varying molecular weights after 17 hours of dialysis. The membrane MWCO is determined as the solute size that is retained by at least 90%. However, since a solute’s permeability is also dependent upon molecular shape, degree of hydration, ionic charge and polarity, we recommend selecting a MWCO that is at least half the size of the MW of the species to be retained and/or twice the size of the MW of the species intended to pass through.

Use only sterile buffers to prepare your membranes before use. This will ensure that microbial contamination will not compromise membrane integrity. Never store hydrated membranes in any buffer without an effective anti-microbial agent e.g. 0.1% sodium azide or 15% EtOH. Never let hydrated membranes dry as that irreversibly changes the pore structure and results in loss of membrane integrity.

We recommend storing them no longer than 4 weeks in 20% ethanol at 4ºC if the initial buffer was sterile. The ethanol is initially added because it helps to remove any glycerin which is added to the membrane during manufacturing to help promote hydration. The key point is that one must avoid any bacterial growth as they may produce cellulases that modify/destroy the membranes.

Volumes from 25μl to 150μl can be used in each side of the dialysis well with a maximum total volume of up to 300ul. Detection sensitivity will often dictate the appropriate volume required.

This is dependent on several factors, incubation temperature, compound structure, and shaking. Most compounds reach equilibrium in less than 6 hours at 37ºC shaking at 80 rpm. We recommend a simple kinetic experiment with compound spiked into buffer and dialyzed against buffer to evaluate the equilibrium time required prior to initiating any binding experiments.

The device is a small bench top, manually operated unit with the following dimensions, 6.7” x 4.6”x 1.5” and weight is 1.2lbs. It does not require any utilities or peripherals and it does not require maintenance or service. The 96-well Teflon block conforms to the SBS standards for 96 well plate well centers.

The HTD96b has a standard SBS 96-well base that is compatible with most robotic workstations. Many users have successfully automated their assays using a variety of commercial workstations, including those from the following manufacturers ApricotDesigns, Tecan, Hamilton, Packard, and Beckman.

Yes radio isotopes are used and standard decontamination procedures using “COUNT-OFF” from Perkin Elmer followed by standard cleaning protocols are recommended. However, care must be taken with the stainless steel pressure plate to avoid rusting the disc springs. If feasible do not wash or soak the pressure plate. Consider the option of lining the device cavity with a thin plastic (Saran wrap or equivalent food sealing plastic wrap). This will protect the entire device and all you need decontaminate is the Teflon block.

After assembling dialysis apparatus following the Operating Instructions:

1. Add 150ul of phosphate buffer or protein free serum to the receiving side of the dialysis well.

2. Add 150ul of serum (pH adjusted to 7.4) spiked with 10uM test compound to the Sample side of the dialysis well.

3. Dialyze for 6 hours

4. ACN precipitate and dilute samples prior to analysis in 1.2ml polypropylene tubes. (see instructions below)

5. Remove 10uL from the sample side of the dialysis well and add to 1.2ml tube containing 90uL of phosphate buffer + 300ul of ACN.

6. Remove 90uL from the buffer side of the dialysis well and add to 1.2ml tube containing 10uL of clean serum + 300uL of ACN.

The samples are then quantitated via the Mass Spec / HPLC. When samples are diluted/extracted in this manner, all samples are in a common matrix and the peak height/area can be directly compared.

All values from the Mass Spec / HPLC are corrected for sample dilution (dilution by ACN is ignored because it is the same for all samples). To correct for dilutions, values from the sample side are multiplied by 10 and values from the buffer side are multiplied by 1.1.

A standard curve can be generated to demonstrate that there is a correlation between peak height/area and compound concentration if desired.

Calculated values:

Fraction unbound = fu = concentration on the buffer side / Concentration on the sample side. These can be peak height or area values corrected by the dilution factor as outlined above.

12-14K MWCO 86%

6-8K MWCO 14%

Other membranes have been used for special applications:

10K WWCO ~3.1%

3.5K MWCO ~2.4%

25K MWCO ~0.4%

50K MWCO ~0.13%

Inadvertently using double membranes in the Teflon block instead of ensuring they are separated after hydration.

Loss of membrane integrity during an experiment will manifest as the presence of proteins in the dialysate and a violation of protein mass balance for the well. This may be caused by microbial contamination and enzymatic degradation of the cellulose membrane. The remedy is to ensure correct membrane preparation and use – see above (Membrane hydration and preparation) and this includes thorough cleaning of the Teflon blocks between uses – (How should I clean the Teflon block). If “leakage” persists, sterilize the Teflon block by autoclaving and repeat the experiment to confirm that the Teflon block was contaminated and caused the leakage.

The sealing of the unit depends on the compression of the Teflon blocks by the pressure plate. The most common cause of leakage after extensive use is micro-striations on the Teflon bars due to inappropriate washing with abrasives or brushes. Replacement of the Teflon block remedies this cause.

After each use the HTD dialysis block should be cleaned thoroughly with a non-ionic detergent. Disassemble the dialysis block and soak overnight in a 2-liter beaker containing a 2% solution of Micro 90 (VWR international, Catalog # 21830-416). Rinse the Teflon bars in the 2-liter beaker followed by holding each Teflon bar under a running distilled water stream. Care must be taken to ensure that all detergent has been rinsed from the unit as residual detergent could compromise future binding studies. The Teflon block may also be disinfected with a 10% v/v solution made using commercial Clorox bleach which is 5.25% sodium hypochlorite. The final solution would this be 0.525% sodium hypochlorite solution. Never use any abrasive or brush for cleaning the Teflon blocks as they will cause micro-striations and prevent effective sealing.

The dry membranes can be stored for up to 2 years in sealed or Ziploc bags at 4ºC or ambient temperature. Storage in such bags prevents membranes from drying out and losing their integrity.